CN106795523B - Method for preparing rebaudioside I and application - Google Patents

Abstract

Methods of preparing rebaudioside I, including highly purified rebaudioside I, are described herein. These methods utilize biocatalysts that convert rebaudioside a to rebaudioside I. Also provided are compositions and consumables comprising rebaudioside I, including sweetener compositions and flavor enhancing compositions.

Description

Method for preparing rebaudioside I and application

Cross Reference to Related Applications

This application claims priority to U.S. patent application No. 62/039,344, filed on 8/19/2014, which is incorporated herein by reference in its entirety.

Technical Field

The present invention relates to a biocatalytic method for preparing rebaudioside I. The invention also relates to the use of rebaudioside I as a sweetener, sweetness enhancer and/or flavor enhancer.

Background

High intensity sweeteners have sweetness levels many times higher than sucrose sweetness levels. They are essentially calorie-free and are commonly used in diet and low calorie products, including foods and beverages. High intensity sweeteners do not elicit a glycemic response, making them suitable for use in products directed to diabetics and other populations interested in controlling their carbohydrate intake.

Steviol glycosides are a class of compounds found in the leaves of Stevia rebaudiana (Bertoni), a perennial shrub of the Asteraceae (Compositae) native to certain areas of south America. They are structurally characterized by a single base, steviol, differing by the presence of carbohydrate residues at positions C13 and C19. They accumulate in the leaves of stevia, constituting approximately 10% -20% of the total dry weight. The 4 major glycosides found in the leaves of stevia on a dry basis typically include stevioside (stevioside) (9.1%), rebaudioside a (3.8%), rebaudioside C (0.6% -1.0%) and dulcoside a (dulcoside a) (0.3%).

WO2010/03911 by Morita Kagaku Kogyo co., Ltd discloses that small amounts of rebaudioside I are found in the leaves of certain stevia species. WO2010/03911 does not describe any organoleptic properties of the isolated rebaudioside I or any composition containing a high concentration of rebaudioside I.

Thus, there remains a need for simple, efficient, and economical methods for preparing and purifying rebaudioside I, particularly when these methods are available on a commercial scale.

Disclosure of Invention

The present invention provides a biocatalytic method for preparing rebaudioside I compositions comprising contacting a starting composition comprising rebaudioside a with a biocatalyst capable of converting rebaudioside a to rebaudioside I.

In one embodiment, the biocatalyst is a UDP-glycosyltransferase (UGT). Suitable UGTs include, but are not limited to UGT76G1 or a variant thereof, wherein the variant comprises at least about 75% amino acid sequence identity to UGT76G 1.

In a particular embodiment, the UGT76G1 variant contains one or more point mutations found to increase rebaudioside a to rebaudioside a and/or rebaudioside D to rebaudioside M conversion by at least about 5% compared to using unmutated UGT76G1 under identical conditions. Suitable mutations include, but are not limited to, Q266E, P272A, R334K, G348P, L379G, S42A, F46I, I190L, S274G, I295M, K303G, F314S, K316R, K393R, V394I, I407V, N409V, Q425V, Q432V, S447V, S456V, I46V, I295V, S119V, S274V, K334V, F314V, K303V, K316V, K190V, I425V, Q432V, N138V, V V, F182V, V407V, a 272V, V V, E393 a 393, and E393 36449.

In a preferred embodiment, the UGT76G1 variant is selected from the group consisting of: UGT76G1-R1-F12, UGT76G1-R2-B9 and UGT76G 1-R3-G3.

The biocatalyst may be provided in any form, such as, for example, in pure form, as a crude lysate, or as a whole cell suspension. In another alternative embodiment, the biocatalyst is provided as a microorganism.

The starting composition may be rebaudioside a, a mixture of steviol glycosides, or a stevia extract in pure form. In one embodiment, the starting composition is a steviol glycoside mixture or stevia extract containing at least about 1% rebaudioside a by weight.

The methods of the present invention provide rebaudioside I compositions comprising at least about 1% rebaudioside I by weight.

The method is typically carried out in a culture medium. Thus, the method can further comprise separating the rebaudioside I composition from the culture medium to provide a separated rebaudioside I composition. The method can also further comprise a purification step wherein the isolated rebaudioside I composition is purified to provide a highly purified rebaudioside I composition comprising at least about 80% rebaudioside I by weight. In one embodiment, the isolated rebaudioside I composition is purified to provide pure rebaudioside I, i.e., > 99% by weight.

The invention also provides pure rebaudioside I.

The present invention also provides compositions comprising rebaudioside I. In one embodiment, the present invention is a composition comprising rebaudioside I in an amount from about 1% to about 99% by weight. In one embodiment, the present invention is a highly purified rebaudioside a composition comprising at least about 80% rebaudioside I by weight.

The invention also includes methods of using rebaudioside I as a sweetener, flavor enhancer, or sweetness enhancer.

The invention also extends to sweetener compositions, flavor enhancing compositions, and sweetness enhancing compositions comprising rebaudioside I.

Consumable products comprising rebaudioside I and compositions comprising rebaudioside I are also provided. Exemplary consumable products include pharmaceutical compositions, edible gel mixes or compositions, dental compositions, confections, flavoring compositions, chewing gum compositions, cereal compositions, baked goods, dairy products, and tabletop (tabletop) sweetener compositions, beverages, or beverage products.

Brief description of the drawings

The accompanying drawings are included to provide a further understanding of the invention. The drawings illustrate embodiments of the invention and, together with the description, serve to explain the principles of embodiments of the invention.

Fig. 1 shows the biocatalytic production of rebaudioside I.

Fig. 2 shows the biocatalytic production of rebaudioside a from stevioside using the enzyme UGT76G1 with concomitant recycling of UDP to UDP-glucose by sucrose synthase.

FIG. 3 shows UGT76G 1-catalyzed conversion of stevioside to rebaudioside A.

FIG. 4 shows the UGT76G1-R1-F12 catalyzed reaction profile of rebaudioside A to rebaudioside I.

Fig. 5 shows the activity of UGT mutants for converting rebaudioside a to rebaudioside I.

FIG. 6 shows an HPLC trace of rebaudioside I before purification.

FIG. 7 shows the sensory attributes of rebaudioside I and rebaudioside M at 400ppm in water at 4 ℃.

Detailed Description

Method for preparing rebaudioside I

The present invention provides a biocatalytic method for preparing rebaudioside I compositions by contacting a starting composition comprising rebaudioside a with a biocatalyst capable of converting rebaudioside a to rebaudioside I. The rebaudioside I composition can optionally be further processed to provide highly purified rebaudioside I or even pure rebaudioside I.

As used herein, "starting composition" refers to any composition comprising rebaudioside a. Typically, the starting composition is an aqueous solution.

The starting composition may be purified rebaudioside a, a mixture of steviol glycosides, or a stevia extract. Steviol glycoside mixtures and stevia extracts containing rebaudioside a, as well as highly purified rebaudioside a, are commercially available from various suppliers or can be readily prepared by methods provided in the literature. For example, U.S. patent No. 8,791,253 to Coca-Cola Company (the contents of which are incorporated herein by reference in their entirety) provides a method for obtaining rebaudioside a in greater than 95% purity.

In one embodiment, the starting composition is purified rebaudioside a, i.e., > 99% by weight.

In another embodiment, the starting composition is a steviol glycoside mixture. The identity of the steviol glycoside mixture is not particularly limited and may include naturally occurring steviol glycosides, e.g., steviolmonoside, steviolbioside, rubusoside, dulcoside B, dulcoside a, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside a, rebaudioside I, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M2, rebaudioside D2, rebaudioside N, rebaudioside O; synthetic steviol glycosides, e.g., enzymatically glycosylated steviol glycosides and combinations thereof.

In some embodiments, the starting composition is a steviol glycoside mixture or a stevia extract that comprises rebaudioside a in an amount of about 1% or more by weight, such as, for example, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, or about 90% or more.

In a particular embodiment, the starting composition is a steviol glycoside mixture or stevia extract comprising rebaudioside a in an amount of about 50% or more by weight.

In another particular embodiment, the starting composition is a steviol glycoside mixture or stevia extract comprising rebaudioside a in an amount of about 80% or more by weight.

In yet another particular embodiment, the starting composition is a steviol glycoside mixture or stevia extract comprising rebaudioside a in an amount of about 90% or more by weight.

In yet another specific embodiment, the starting composition is a steviol glycoside mixture or stevia extract comprising rebaudioside a in an amount of about 95% or more, such as, for example, about 96%, about 97%, about 98%, or about 99% by weight.

Notably, the starting composition can comprise an amount of rebaudioside I. For example, the starting composition is a stevia extract or a mixture of steviol glycosides. Contacting the biocatalyst with the starting composition produces a rebaudioside I composition that contains an increased amount of rebaudioside I compared to the amount of rebaudioside I (if any) present in the starting composition. The increase in rebaudioside I was attributed to the action of the biocatalyst.

In a particular embodiment, the amount of rebaudioside I in the rebaudioside composition (i.e., the composition resulting from the method of this invention) is about 0.5, about 1, about 3, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60 or about 65, about 70 or about 75% or more greater than the amount of rebaudioside I present in the starting composition.

The starting composition is contacted with the biocatalyst in an aqueous medium comprising water and, for example, various components selected from the group consisting of carbon sources, energy sources, nitrogen sources, trace elements, vitamins, nucleosides, nucleoside phosphates, nucleoside diphosphates, nucleoside triphosphates, organic and inorganic salts, organic and mineral acids, bases, and the like. The carbon source includes glycerol, glucose, carbon dioxide, carbonate, and bicarbonate. The nitrogen source may include nitrate, nitrite, amino acids, peptides, peptones, or proteins.

In a particular embodiment, the medium comprises a buffer. Suitable buffers include, but are not limited to, PIPES buffer, acetate buffer, and phosphate buffer. In a particular embodiment, the medium comprises a phosphate buffer.

In one embodiment, the medium may also include an organic solvent, such as methanol, ethanol, propanol, and the like.

As used herein, "biocatalyst" refers to an enzyme capable of converting rebaudioside a to rebaudioside I. The enzyme may be a naturally occurring or recombinant protein. At least one biocatalyst is used in the present method for converting rebaudioside a to rebaudioside I. However, if necessary, a variety of biocatalysts may be used. Thus, in some embodiments, two or more biocatalysts are used, such as, for example, three or more biocatalysts, four or more biocatalysts, or five or more biocatalysts.

The biocatalyst may be provided in the form of a whole cell suspension, a crude lysate, a purified enzyme, or a combination thereof. In one embodiment, the biocatalyst is provided in purified form, i.e., as a purified enzyme. In another embodiment, the biocatalyst is provided in the form of a crude lysate. In yet another embodiment, the biocatalyst is provided in the form of a whole cell suspension.

In another embodiment, the biocatalyst is provided in the form of one or more cells, i.e., the biocatalyst is associated with one or more cells. The biocatalyst may be located on the cell surface, inside the cell, or both on the cell surface and inside the cell.

In another embodiment, the biocatalyst is provided in the form of a microorganism, i.e., the biocatalyst is associated with the microorganism. The microorganism can be any microorganism having one or more necessary biocatalysts/enzymes for the conversion of rebaudioside a to rebaudioside I. Suitable microorganisms include, but are not limited to, Escherichia coli, Saccharomyces species, Aspergillus species, Pichia species, Bacillus species, yarrowia species, and the like.

In one embodiment, the microorganism is free (i.e., not immobilized) when contacted with the starting composition.

In another embodiment, the microorganism is immobilized when contacted with the starting composition. For example, the microorganism may be immobilized to a solid support made of inorganic or organic material. Non-limiting examples of solid supports suitable for immobilizing microorganisms include derivatized cellulose or glass, ceramics, metal oxides, or membranes. The microorganism may be immobilized to the solid support, for example by covalent attachment, adsorption, cross-linking, capture or encapsulation.

In yet another embodiment, the biocatalyst is secreted into the reaction medium by the microorganism.

Suitable biocatalysts for the conversion of rebaudioside a to rebaudioside I include, but are not limited to, steviol biosynthetic enzymes and UDP-glycosyltransferase (UGT).

In one embodiment, the biocatalyst is a steviol biosynthetic enzyme, e.g., a Mevalonate (MVA) pathway enzyme.

In another embodiment, the biocatalyst is a steviol biosynthetic enzyme, e.g., a non-mevalonate 2-C-methyl-D-erythritol 4-phosphate pathway (MEP/DOXP) enzyme.

In one embodiment, the biocatalyst is a steviol biosynthetic enzyme selected from the group consisting of: geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, kaurene oxidase, kaurenoic acid 13-hydroxylase (KAH), steviol synthase, deoxyxylulose 5-phosphate synthase (DXS), D-1-deoxyxylulose 5-phosphate reductoisomerase (DXR), cytidine-2-C-methyl-D-erythritol synthase (CMS) 4-diphosphate, cytidine-2-C-methyl-D-erythritol kinase (CMK), cytidine-2-C-methyl-D-erythritol kinase 4-diphosphate (MCS), l-hydroxy-2-methyl-2 (E) -butenyl-4-diphosphate synthase (HDS), l-hydroxy-2-methyl-2 (E) -butenyl 4-diphosphate reductase (HDR), acetoacetyl-CoA thiolase, truncated HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, and cytochrome P450 reductase.

In one embodiment, the biocatalyst is a UGT capable of adding at least one glucose unit to rebaudioside a to provide rebaudioside I. In one embodiment, the UGT is UGT76G1 or a variant thereof, wherein the variant comprises about 75% or greater amino acid sequence identity to UGT76G 1. Exemplary UGT76G1 variants include, but are not limited to, UGTSL2, UGTLB, UGT91D2, UGT76G1-R1-F12, UGT76G1-R2-B9, and UGT76G 1-R3-G3.

In another embodiment, the biocatalyst is UGT selected from the list of genetic information (GenInfo) identifiers, preferably from the group presented in table 1, and more preferably the group presented in table 2.

TABLE 1

TABLE 2

GI number	Login number	Origin of origin	Internal standard
				460409128	XP.004249992.1	Tomato	UGTSL
460386018	XP.004238697.1	Tomato	-
				460409134	XP.004249995.1	Tomato	-
460410132	XP.004250485.1	Tomato	UGTSL2
				460410130	XP.004250484.1	Tomato	-
460410128	XP.004250483.1	Tomato	-
				460378310	XP.004234916.1	Tomato	-
209954733	BAG80557.1	Ningxia matrimony vine	UGTLB
				209954725	BAG80553.1	Ningxia matrimony vine	-

In one embodiment, the biocatalyst is a UGT76G1 variant comprising one or more point mutations found to increase rebaudioside a to rebaudioside I conversion by at least about 5% (wherein the results are normalized) compared to using unmutated UGT76G1 under the same conditions. In another embodiment, the biocatalyst is a UGT76G1 variant comprising one or more point mutations found to increase rebaudioside D to rebaudioside M conversion by at least about 5% (wherein the results are normalized) compared to using unmutated UGT76G1 under the same conditions.

In one embodiment, the biocatalyst is a purified UGT76G1 variant, i.e., UGT76G1 provided in the form of a purified enzyme. In another embodiment, the biocatalyst is a UGT76G1 variant provided in the form of a crude lysate. In yet another embodiment, the biocatalyst is a UGT76G1 variant provided in the form of a whole cell suspension.

In a particular embodiment, the biocatalyst is purified UGT76G1 comprising one or more of the following point mutations: Q266E, P272A, R334K, G348P and L379G. In a more specific embodiment, the biocatalyst is UGT76G1 comprising all of the following mutations: Q266E, P272A, R334K, G348P and L379G, i.e. the biocatalyst is UGT76G 1-R1-F12. In a specific embodiment, the biocatalyst is UGT76G1-R1-F12 provided in the form of a purified enzyme. In another embodiment, the biocatalyst is UGT76G1-R1-F12 provided in the form of a crude lysate. In yet another embodiment, the biocatalyst is UGT76G1-R1-F12 provided in the form of a whole cell suspension.

In another specific embodiment, the biocatalyst is UGT76G1 comprising one or more of the following point mutations: Q266E, P272A, R334K, G348P, L379G, S42A, F46I, I190L, S274G, I295M, K303G, F314S, K316R, K393R, V394I, I407V, N409K, N409R, Q425E, Q432E, S447A and S456L. In a more specific embodiment, the biocatalyst is a UGT76G1 variant comprising all of the above mutations, i.e., the biocatalyst is UGT76G 1-R2-B9. In a particular embodiment, the biocatalyst is purified UGT76G1-R2-B9, i.e., UGT76G1-R2-B9 provided in the form of a purified enzyme. In another embodiment, the biocatalyst is UGT76G1-R2-B9 provided in the form of a lysate. In yet another embodiment, the biocatalyst is UGT76G1-R2-B9 provided in the form of a whole cell suspension.

In yet another embodiment, the biocatalyst is a UGT76G1 variant comprising one or more of the following point mutations: q266, P272, R334, G348, L379, S42, F46, I190, S274, I295, K303, F314, K316, K393, V394, I407, N409, Q425, Q432, S447, S456, I46, I295, S119, S274, K334, F314, K303, K316, K393, I190, Q425, Q432, N138, V394, F182, V407, a272, V264, E449, and a 352. In a more specific embodiment, the biocatalyst is a UGT76G1 variant comprising all of the above mutations, i.e., the biocatalyst is UGT76G 1-R3-G3. In a specific embodiment, the biocatalyst is purified UGT76G1-R3-G3, i.e., UGT76G1-R3-G3 provided in the form of a purified enzyme. In another embodiment, the biocatalyst is UGT76G1-R3-G3 provided in the form of a lysate. In yet another embodiment, the biocatalyst is UGT76G1-R3-G3 provided in the form of a whole cell suspension.

Using the UGT76G1 variant as a biocatalyst in the methods of the invention results in at least about a 5% increase in rebaudioside a to rebaudioside I conversion compared to using unmutated UGT76G1 under the same conditions (wherein the results are normalized). In preferred embodiments, the conversion is increased from about 5% to about 1,000,000%, such as, for example, from about 5% to about 100,000%, from about 5% to about 10,000%, from about 5% to about 1,000%, from about 5% to about 500%, from about 5% to about 250%, from about 5% to about 100%, from about 50%, from about 20% to about 50%, from about 30% to about 50%, or from about 40% to about 50%.

Optionally, the process of the invention further comprises recycling UDP to provide UDP-glucose. Accordingly, the methods include concomitantly recycling UDP by providing a recycle catalyst (i.e., a catalyst capable of overproducing UDP-glucose) and a recycle substrate to effect conversion of rebaudioside a to rebaudioside I using catalytic amounts of UDP-glucosyltransferase and UDP-glucose (fig. 2).

In one embodiment, the UDP-glucose recycling catalyst is a sucrose synthase and the recycling substrate is sucrose.

In a particular embodiment, the methods of the present invention provide rebaudioside I compositions comprising rebaudioside I in an amount of about 1% or more, such as, for example, about 5% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, or about 90% or more, by weight.

In a particular embodiment, the method provides a composition comprising rebaudioside I in an amount of about 50% or more by weight.

In another particular embodiment, the method provides a composition comprising rebaudioside I in an amount of about 80% or more by weight.

In yet another particular embodiment, the method provides a composition comprising rebaudioside I in an amount of about 90% or more by weight.

In yet another particular embodiment, the method provides a composition comprising rebaudioside I in an amount of about 95% by weight or greater, such as, for example, about 96%, about 97%, about 98%, or about 99% by weight.

Optionally, the methods of the invention further comprise separating rebaudioside I from the culture medium. Any suitable method of separation may be used, such as, for example, lysis, crystallization, separation by membrane, centrifugation, extraction (liquid or solid phase), chromatographic separation, HPLC (preparative or analytical) or a combination of such methods. In a specific embodiment, the separation can be achieved by lysis and centrifugation.

In one embodiment, rebaudioside I is continuously removed from the medium while conversion progresses. In another embodiment, rebaudioside I is isolated and optionally purified from the culture medium after the reaction is complete.

Isolation from the culture medium may yield a composition having a rebaudioside I content that is lower than desired and/or the composition may comprise additional components, such as undesired steviol glycosides (in identity or content) and/or residual reaction products. Thus, the composition can be further purified to provide a highly purified rebaudioside I composition. The term "highly purified" as used herein refers to compositions having greater than about 80% rebaudioside I by weight on a dry basis. In one embodiment, the highly purified rebaudioside I composition comprises greater than about 90% rebaudioside I by weight, such as, for example, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, or about 99% rebaudioside I by weight. In exemplary embodiments, the composition can be further purified to provide pure rebaudioside I, i.e., > 99% rebaudioside I by weight on a dry basis.

Purification may be effected by any means known to those skilled in the art including, but not limited to, crystallization, separation by membrane, centrifugation, extraction (liquid or solid phase), chromatographic separation, HPLC (preparative or analytical) or a combination of such methods. In a particular embodiment, rebaudioside I is purified using HPLC. In a more particular embodiment, rebaudioside I is purified using semi-preparative HPLC.

In one embodiment, the present invention provides a method for preparing a rebaudioside I composition comprising (a) contacting a starting composition comprising rebaudioside a with an enzyme capable of converting rebaudioside a to rebaudioside I to provide a composition comprising rebaudioside I and (b) separating rebaudioside I to provide a rebaudioside I composition (i.e., a separated rebaudioside I composition).

In a more particular embodiment, the present invention provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a starting composition comprising rebaudioside a with UGT76G1 or a variant thereof having about 75% or more amino acid sequence identity, and UDP glucose to form a composition comprising rebaudioside I, and (b) separating rebaudioside I to provide a separated rebaudioside I composition. Exemplary UGT variants include, but are not limited to, UGT76G1-R1-F12, UGT76G1-R2-B9, and UGT76G 1-R3-G3. Optionally, the method comprises concomitant UDP-glucose recycling by providing sucrose synthase and sucrose in (a).

In another more particular embodiment, the present invention provides a method for preparing a rebaudioside I composition comprising (a) contacting a medium containing a starting composition comprising rebaudioside a with at least UGT76G1 or a variant thereof having about 75% or more amino acid sequence identity thereto and UDP-glucose to form a composition comprising rebaudioside I, and (b) separating rebaudioside I from the medium to provide a separated rebaudioside I composition. Exemplary UGT variants include, but are not limited to, UGT76G1-R1-F12, UGT76G1-R2-B9, and UGT76G 1-R3-G3. Optionally, the method comprises concomitant UDP-glucose recycling by providing sucrose synthase and sucrose in (a).

In another embodiment, the present invention provides a method for preparing a rebaudioside I composition comprising (a) contacting a starting composition comprising rebaudioside a with an enzyme capable of converting rebaudioside a to rebaudioside I to provide a composition comprising rebaudioside I, (b) separating rebaudioside I to provide a separated rebaudioside I composition, and (c) purifying the separated rebaudioside I composition to provide a highly purified rebaudioside I composition.

In yet another more particular embodiment, the present invention provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a starting composition comprising rebaudioside a with UGT76G1 or a variant thereof having about 75% or more amino acid sequence identity, and UDP-glucose to form a composition comprising rebaudioside I, (b) separating rebaudioside I to provide a separated rebaudioside I composition, and (c) purifying the separated rebaudioside I composition to provide a highly purified rebaudioside I composition. Optionally, the method comprises concomitant UDP-glucose recycling by providing sucrose synthase and sucrose in (a). Exemplary UGT variants include, but are not limited to, UGT76G1-R1-F12, UGT76G1-R2-B9, and UGT76G 1-R3-G3.

In another more particular embodiment, the present invention provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a medium containing a starting composition comprising rebaudioside a with UGT76G1 or a variant thereof having about 75% or more amino acid sequence identity, to provide a composition comprising rebaudioside I, (b) separating rebaudioside I from the medium to provide a separated rebaudioside I composition, and (c) purifying the separated rebaudioside I composition to provide a highly purified rebaudioside I composition. Optionally, the method comprises concomitant UDP-glucose recycling by providing sucrose synthase and sucrose in (a). Exemplary UGT variants include, but are not limited to, UGT76G1-R1-F12, UGT76G1-R2-B9, and UGT76G 1-R3-G3.

In another embodiment, the present invention provides a method for preparing a highly purified rebaudioside I composition comprising (a) contacting a starting composition comprising rebaudioside a with an enzyme capable of converting rebaudioside a to rebaudioside I to provide a rebaudioside I comprising composition and b) purifying the rebaudioside I comprising composition to provide a highly purified rebaudioside I composition.

In yet another particular embodiment, the present invention provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a starting composition comprising rebaudioside a with UGT76G1 or a variant thereof having about 75% or more amino acid sequence identity, and UDP-glucose to form a rebaudioside I comprising composition, and (b) purifying the rebaudioside I comprising composition to provide a highly purified rebaudioside I composition. Optionally, the method comprises concomitant UDP-glucose recycling by providing sucrose synthase and sucrose in (a). Exemplary UGT variants include, but are not limited to, UGT76G1-R1-F12, UGT76G1-R2-B9, and UGT76G 1-R3-G3.

In yet another particular embodiment, the present invention provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a medium containing a starting composition comprising rebaudioside a with UGT76G1 or a variant thereof having about 75% or more amino acid sequence identity, and UDP-glucose to form a composition comprising rebaudioside I, and (b) purifying the composition comprising rebaudioside I to provide a highly purified rebaudioside I composition. Optionally, the method comprises concomitant UDP-glucose recycling by providing sucrose synthase and sucrose in (a). Exemplary UGT variants include, but are not limited to, UGT76G1-R1-F12, UGT76G1-R2-B9, and UGT76G 1-R3-G3.

Fermentation can also be used to resynthesize rebaudioside I. For example, a method of producing a rebaudioside I composition, the method comprising (a) contacting glucose with a microorganism comprising at least one enzyme capable of converting glucose to rebaudioside I to provide a rebaudioside I composition, and (b) separating rebaudioside I to provide a separated rebaudioside I composition. Optionally, the method further comprises purifying rebaudioside I to provide highly purified rebaudioside I.

The fermentation and biocatalytic steps may be used sequentially. For example, fermentation of a composition comprising glucose with a microorganism containing at least one enzyme capable of converting glucose to the target steviol glycoside (e.g., rebaudioside a) can be performed first. The target steviol glycoside, e.g., rebaudioside a, which now becomes the starting material for the next bioconversion purpose, can then be contacted with a biocatalyst capable of converting it to the next target steviol glycoside, e.g., rebaudioside I.

Between each conversion, the steviol glycoside of interest may optionally be separated from the culture medium before contacting with the next biocatalyst.

In one embodiment, rebaudioside a of the starting composition of the present method is prepared by contacting stevioside with an enzyme capable of converting stevioside to rebaudioside a. In a particular embodiment, the enzyme is any UDP-glucosyltransferase capable of adding at least one glucose unit thereto, thereby producing rebaudioside a. The UDP-glucosyltransferase can be, for example, UGT76G 1.

Accordingly, the present invention provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a composition comprising stevioside with an enzyme capable of converting stevioside to rebaudioside a to provide a composition comprising rebaudioside a, (b) separating rebaudioside a, (c) contacting a composition comprising rebaudioside a with an enzyme capable of converting rebaudioside a to rebaudioside I to provide a composition comprising rebaudioside I, and (d) separating rebaudioside I to provide a separated rebaudioside I composition. The method can further comprise purifying the separated rebaudioside I composition to provide highly purified rebaudioside I.

In a more particular embodiment, the present invention provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a stevioside-containing composition with UGT76G1 and UDP-glucose to provide a rebaudioside a-containing composition, (b) separating rebaudioside a, (c) contacting the rebaudioside a-containing composition with UGT76G1 or a variant thereof having about 75% or more amino acid sequence identity and UDP-glucose to provide a rebaudioside I-containing composition, and (d) separating rebaudioside I to provide a separated rebaudioside I composition. The method can further comprise purifying the separated rebaudioside I composition to provide a highly purified rebaudioside I composition. Optionally, the process comprises concomitant UDP-glucose recycling in one or both contacting steps by providing sucrose synthase and sucrose. Exemplary UGT variants include, but are not limited to, UGT76G1-R1-F12, UGT76G1-R2-B9, and UGT76G 1-R3-G3.

In another more particular embodiment, the invention provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a medium containing a composition comprising stevioside with UGT76G1 and UDP-glucose to provide a composition comprising rebaudioside a, (b) isolating rebaudioside a from the medium, (c) contacting a medium containing a composition comprising rebaudioside a with UGT76G1 or a variant thereof having about 75% or greater amino acid sequence identity and UDP-glucose to provide a composition comprising rebaudioside I, and (d) separating rebaudioside I from the medium to provide an isolated rebaudioside I composition. The method can further comprise purifying the separated rebaudioside I composition to provide a highly purified rebaudioside I composition. Optionally, the process comprises concomitant UDP-glucose recycling in one or both contacting steps by providing sucrose synthase and sucrose. Exemplary UGT variants include, but are not limited to, UGT76G1-R1-F12, UGT76G1-R2-B9, and UGT76G 1-R3-G3.

In another embodiment, the invention provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a composition comprising glucose with a microorganism comprising at least one enzyme capable of converting glucose to rebaudioside a to provide a composition comprising rebaudioside a, (b) separating rebaudioside a, (c) contacting the composition comprising rebaudioside a with an enzyme capable of converting rebaudioside a to rebaudioside I to provide a composition comprising rebaudioside I, and (d) separating rebaudioside I from the culture medium to provide a separated rebaudioside I composition. The method can further comprise purifying the separated rebaudioside I composition to provide a highly purified rebaudioside I composition.

In a more particular embodiment, the present invention provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a composition comprising glucose with a microorganism comprising at least one enzyme capable of converting glucose to rebaudioside a to provide a composition comprising rebaudioside a, (b) separating rebaudioside a, (c) contacting the composition comprising rebaudioside a with UGT76G1 or a variant thereof having about 75% or more amino acid sequence identity and UDP-glucose to provide a composition comprising rebaudioside I, and (d) separating rebaudioside I to provide a separated rebaudioside I composition. The method can further comprise purifying the separated rebaudioside I composition to provide highly purified rebaudioside I. Optionally, the process comprises concomitant UDP-glucose recycling in one or both contacting steps by providing sucrose synthase and sucrose. Exemplary UGT variants include, but are not limited to, UGT76G1-R1-F12, UGT76G1-R2-B9, and UGT76G 1-R3-G3.

In another more particular embodiment, the invention provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a medium comprising a composition comprising glucose with a microorganism comprising at least one enzyme capable of converting glucose to rebaudioside a to provide a composition comprising rebaudioside a, (b) isolating rebaudioside a from the medium, (c) contacting the medium comprising the composition comprising rebaudioside a with UGT76G1 or a variant thereof having about 75% or more amino acid sequence identity and UDP-glucose to provide a composition comprising rebaudioside I, and (d) isolating rebaudioside I from the medium to provide an isolated rebaudioside I composition. The method can further comprise purifying the separated rebaudioside I composition to provide highly purified rebaudioside I. Optionally, the process comprises concomitant UDP-glucose recycling in one or both contacting steps by providing sucrose synthase and sucrose. Exemplary UGT variants include, but are not limited to, UGT76G1-R1-F12, UGT76G1-R2-B9, and UGT76G 1-R3-G3.

In one embodiment, the stevioside is prepared by contacting rubusoside with an enzyme capable of converting rubusoside to stevioside. In one embodiment, the enzyme is any UDP-glucosyltransferase capable of adding at least one glucose unit to rubusoside thereby producing stevioside. The UDP-glucosyltransferase can be, for example, UGT91D2 or a variant thereof having about 75% or more amino acid sequence identity. Exemplary UGT91D2 variants include, but are not limited to, UGTSL and UGTSL 2.

Accordingly, the present invention provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a composition comprising rubusoside with an enzyme capable of converting rubusoside to stevioside, (b) isolating the stevioside, (c) contacting the composition comprising stevioside with an enzyme capable of converting stevioside to rebaudioside a to provide a composition comprising rebaudioside a, (d) isolating rebaudioside a, (e) contacting the composition comprising rebaudioside a with an enzyme capable of converting rebaudioside a to rebaudioside I to provide a composition comprising rebaudioside I, and (d) isolating rebaudioside I to provide an isolated rebaudioside I composition. The method can further comprise purifying the separated rebaudioside I composition to provide highly purified rebaudioside I.

In a more particular embodiment, the present invention provides a method for preparing a rebaudioside I composition, the method comprises (a) contacting a composition comprising rebaudioside with UGT91D2 or a variant thereof having about 75% or more amino acid sequence identity and UDP-glucose, to provide a composition comprising stevioside, (b) isolating the stevioside, (c) contacting the stevioside-containing composition with UGT76G1 and UDP-glucose to provide a rebaudioside a-containing composition, (d) isolating the rebaudioside a, (e) contacting the rebaudioside a-containing composition with UGT76G1 or a variant thereof having about 75% or greater amino acid sequence identity and UDP-glucose to provide a rebaudioside I-containing composition, and (f) isolating the rebaudioside I to provide an isolated rebaudioside I composition. The method can further comprise purifying the separated rebaudioside I composition to provide a highly purified rebaudioside I composition. Optionally, the method comprises concomitant UDP-glucose recycling in any or all of the contacting steps by providing sucrose synthase and sucrose. Exemplary UGT91D2 variants include, but are not limited to, UGTSL and UGTSL 2. Exemplary UGT76G1 variants include, but are not limited to, UGT76G1-R1-F12, UGT76G1-R2-B9, and UGT76G 1-R3-G3.

In another more particular embodiment, the invention provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a medium containing a composition comprising rebaudioside with UGT91D2 or a variant thereof having about 75% or more amino acid sequence identity, and UDP-glucose to provide a composition comprising stevioside, (b) isolating stevioside from the medium, (c) contacting the medium containing the composition comprising stevioside with UGT76G1 and UDP-glucose to provide a composition comprising rebaudioside a, (D) separating rebaudioside a from the medium, (e) contacting the medium containing the composition comprising rebaudioside a with UGT76G1 or a variant thereof having about 75% or more amino acid sequence identity, and UDP-glucose to provide a composition comprising rebaudioside I, and (f) isolating rebaudioside I from the culture medium. The method can further comprise purifying the separated rebaudioside I composition to provide highly purified rebaudioside I. Optionally, the method comprises concomitant UDP-glucose recycling in any or all of the contacting steps by providing sucrose synthase and sucrose. Exemplary UGT91D2 variants include, but are not limited to, UGTSL and UGTSL 2. Exemplary UGT76G1 variants include, but are not limited to, UGT76G1-R1-F12, UGT76G1-R2-B9, and UGT76G 1-R3-G3.

The present invention also provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a composition comprising glucose with a microorganism capable of converting glucose to rebaudioside, (b) isolating rebaudioside, (c) contacting the composition comprising rebaudioside with an enzyme capable of converting rebaudioside to stevioside, to provide a composition comprising stevioside, (d) isolating the stevioside, (e) contacting the stevioside-containing composition with an enzyme capable of converting stevioside to rebaudioside A, to provide a rebaudioside A containing composition, (f) separating rebaudioside A, (g) contacting the rebaudioside A containing composition with an enzyme capable of converting rebaudioside A to rebaudioside I, to provide a composition comprising rebaudioside I, and (h) isolating rebaudioside I. The method can further comprise purifying the separated rebaudioside I composition to provide highly purified rebaudioside I.

In a more particular embodiment, the present invention also provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a composition comprising glucose with a microorganism capable of converting glucose to rebaudioside, (b) isolating rebaudioside, (c) contacting the composition comprising rebaudioside with UGT91D2 or a variant thereof having about 75% or more amino acid sequence identity, and UDP-glucose to provide a composition comprising stevioside, (D) isolating stevioside, (e) contacting the composition comprising rebaudioside with UGT76G1 and UDP-glucose to provide a composition comprising rebaudioside a, (f) isolating rebaudioside a, (G) contacting the composition comprising rebaudioside a with UGT76G1 or a variant thereof having about 75% or more amino acid sequence identity, and UDP-glucose, to provide a composition comprising rebaudioside I, and (h) separating rebaudioside I to provide a separated rebaudioside I composition. The method can further comprise purifying the separated rebaudioside I composition to provide highly purified rebaudioside I. Optionally, the method comprises concomitant UDP-glucose recycling in one, both, or all of the contacting steps by providing sucrose synthase and sucrose. Exemplary UGT91D2 variants include, but are not limited to, UGTSL and UGTSL 2. Exemplary UGT76G1 variants include, but are not limited to, UGT76G1-R1-F12, UGT76G1-R2-B9, and UGT76G 1-R3-G3.

In another more particular embodiment, the invention also provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a medium containing a composition comprising glucose with a microorganism capable of converting glucose to rubusoside, (b) isolating rubusoside from the medium, (c) contacting the medium containing the composition comprising rubusoside with UGT91D2 or a variant thereof having about 75% or more amino acid sequence identity and UDP-glucose to provide a composition comprising stevioside, (D) isolating stevioside from the medium, (e) contacting the medium containing the composition comprising steviol glycoside with UGT76G1 and UDP-glucose to provide a composition comprising rebaudioside a, (f) isolating rebaudioside a from the medium, (G) contacting the medium containing the composition comprising rebaudioside a with UGT76G1 or a variant thereof having an amino acid of about 75% or more A variant of amino acid sequence identity, and UDP-glucose to provide a composition comprising rebaudioside I, and (h) isolating rebaudioside I from the culture medium to provide an isolated rebaudioside I composition. The method can further comprise purifying the separated rebaudioside I composition to provide highly purified rebaudioside I. Optionally, the method comprises concomitant UDP-glucose recycling in any or all of the contacting steps by providing sucrose synthase and sucrose. Exemplary UGT91D2 variants include, but are not limited to, UGTSL and UGTSL 2. Exemplary UGT76G1 variants include, but are not limited to, UGT76G1-R1-F12, UGT76G1-R2-B9, and UGT76G 1-R3-G3.

Compounds and compositions

The present invention provides rebaudioside I having the formula:

13- [ (2-O-beta-D-glucopyranosyl-3-O-beta-D-glucopyranosyl) oxy ] enantiomorph-kauri-16-en-19-oic acid- [ (3-O-beta-D-glucopyranosyl) ester ] (rebaudioside I)

In exemplary embodiments, rebaudioside I may be isolated and purified (i.e., > 99% rebaudioside I by weight on a dry basis) or isolated and highly purified (i.e., greater than about 80% by weight on a dry basis).

The present invention includes compositions, particularly consumable products, comprising rebaudioside I.

In one embodiment, the composition comprises rebaudioside I provided as part of a mixture. In a particular embodiment, the mixture is selected from the group consisting of mixtures of: steviol glycosides, stevia extracts, by-products of other steviol glycoside isolation and purification processes, or any combination thereof. In one embodiment, the mixture comprises rebaudioside I in an amount ranging from about 1% to about 99% by weight on a dry basis, such as, for example, from about 2% to about 99%, from about 3% to about 99%, from about 4% to about 99%, from about 5% to about 99%, from about 10% to about 99%, from about 20% to about 99%, from about 30% to about 99%, from about 40% to about 99%, from about 50% to about 99%, from about 60% to about 99%, from about 70% to about 99%, from about 80% to about 99%, and from about 90% to about 99%, on a dry basis. In particular embodiments, the mixture comprises rebaudioside I in an amount greater than about 90%, e.g., greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, and greater than about 99% by weight on a dry basis.

In one embodiment, the composition comprises rebaudioside I provided in the form of a stevia extract. The stevia extract comprises one or more additional steviol glycosides, including but not limited to naturally occurring steviol glycosides such as steviolmonoside, steviolbioside, rubusoside, dulcoside B, dulcoside a, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside a, rebaudioside I, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M2, rebaudioside D2, rebaudioside N, rebaudioside O, synthetic steviol glycosides such as enzymatically glycosylated steviol glycosides and combinations thereof.

In yet another embodiment, the present invention provides rebaudioside I as a pure compound (i.e., > 99% purity on a dry basis).

Rebaudioside I can be present in the composition in an amount effective to provide the following concentrations when the composition is added to a consumable: from about 1ppm to about 10,000ppm, such as, for example, from about 5ppm to about 10,000ppm, from about 10ppm to about 10,000ppm, from about 15ppm to about 10,000ppm, from about 20ppm to about 10,000ppm, from about 25ppm to about 10,000ppm, from about 50ppm to about 10,000ppm, from about 100ppm to about 10,000ppm, from about 200ppm to about 10,000ppm, from about 300ppm to about 10,000ppm, from about 400ppm to about 10,000ppm, from about 500ppm to about 10,000ppm, from about 600ppm to about 10,000ppm, from about 700ppm to about 10,000ppm, from about 800 to about 10,000ppm, from about 900ppm to about 10,000ppm, from about 1,000ppm to about 10,000ppm, from about 2,000ppm to about 10,000ppm, from about 3,000ppm to about 10,000ppm, from about 4,000ppm to about 5,000 ppm.

In another embodiment, rebaudioside I is present in the composition in an amount effective to provide the following concentrations when the composition is added to a consumable: from about 10ppm to about 1,000ppm, such as, for example, from about 10ppm to about 800ppm, from about 50ppm to about 600ppm or from about 200ppm to about 250 ppm. In a particular embodiment, rebaudioside I is present in the composition in an amount effective to provide a concentration from about 300ppm to about 600ppm when the composition is added to a consumable.

In a particular embodiment, rebaudioside I is present in the composition in an amount effective to provide the following concentrations when the composition is added to a consumable: from about 50ppm to about 800ppm, such as, for example, from about 50ppm to about 100ppm, from about 100ppm to about 150ppm, from about 200ppm to about 250ppm, from about 250ppm to about 300ppm, from about 300ppm to about 350ppm, from about 350ppm to about 400ppm, from about 400ppm to about 450ppm, from about 450ppm to about 500ppm, from about 500ppm to about 550ppm, from about 550ppm to about 600ppm, from about 600ppm to about 650ppm, from about 650ppm to about 700ppm, from about 700ppm to about 750ppm or from about 750ppm to about 800 ppm.

In an exemplary embodiment, rebaudioside I is present in the composition in an amount effective to provide a concentration between about 200ppm and about 300ppm when the composition is added to a beverage.

In an exemplary embodiment, rebaudioside I is present in the composition in an amount effective to provide a concentration between about 500ppm and about 600ppm when the composition is added to a beverage.

In another particular embodiment, rebaudioside I is present in the composition in an amount effective to provide the following concentrations when the composition is added to a consumable: about 50ppm, about 100ppm, about 150ppm, about 200ppm, about 250ppm, about 300ppm, about 350ppm, about 400 ppm, about 450ppm, about 500ppm, about 550ppm, about 600ppm, about 650ppm, about 700ppm, about 750ppm, or about 800 ppm.

In an exemplary embodiment, rebaudioside I is present in the composition in an amount effective to provide a concentration of about 200ppm when the composition is added to a beverage.

In an exemplary embodiment, rebaudioside I is present in the composition in an amount effective to provide a concentration of about 275ppm when the composition is added to a beverage.

In an exemplary embodiment, rebaudioside I is present in the composition in an amount effective to provide a concentration of about 550ppm when the composition is added to a beverage.

In an exemplary embodiment, rebaudioside I is present in the composition in an amount effective to provide a concentration of about 600ppm when the composition is added to a beverage.

In exemplary embodiments, the isolated and purified rebaudioside I, or compositions comprising rebaudioside I, exhibit reduced sweetness linger intensity compared to rebaudioside M. In a particular embodiment, the isolated and purified rebaudioside I or a composition comprising rebaudioside I exhibits a sweetness linger intensity that is reduced by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% over rebaudioside M.

Sweetener composition

In one embodiment, the invention is a sweetener composition comprising rebaudioside I. In a more particular embodiment, the present invention is a sweetener composition comprising highly purified or pure rebaudioside I.

As used herein, "sweetener composition" refers to a composition that can be used to sweeten a sweetenable composition (i.e., a composition that can be sweetened) comprising at least one sweet-tasting component in combination with at least one other material.

In one embodiment, rebaudioside I is the sole sweetener in the sweetener composition, i.e., rebaudioside I is the only compound present in the sweetener composition that provides detectable sweetness. In another embodiment, the sweetener composition comprises a rebaudioside I compound in combination with one or more sweetener compounds.

In exemplary embodiments, the sweetener composition comprises rebaudioside I and a compound selected from the group consisting of: rebaudioside A, B, C, D, E, M, N, O, M2, D2, glycosylated steviol glycoside, mogroside V, erythritol, psicose, stevia extract, luo han guo extract, and combinations thereof.

The amount of rebaudioside I in the sweetener composition can vary. In one embodiment, rebaudioside I is present in the sweetener composition in any amount that imparts the desired sweetness when the sweetener composition is added to a sweetenable composition or a sweetenable consumable.

The sweetness of a non-sucrose sweetener can also be measured relative to a sucrose reference by determining the sucrose equivalent of the non-sucrose sweetener. Typically, a trained taster measures the sweetness of a reference sucrose solution containing between 1% -15% sucrose (w/v). Other non-sucrose sweeteners were then tasted at a series of dilutions to determine the concentration of non-sucrose sweetener that was as sweet as the given percentage of sucrose reference. For example, if a 1% solution of a sweetener is as sweet as a 10% sucrose solution, the sweetener is said to be 10 times as potent as sucrose.

In one embodiment, rebaudioside I is present in the sweetener composition in an amount effective to provide greater than about 10% (w/v) sucrose equivalents when added to a sweetener composition or sweetened consumable, such as, for example, greater than about 11%, greater than about 12%, greater than about 13%, or greater than about 14%.

The amount of sucrose in one reference solution, and thus another measure of sweetness, can be described in degrees brix (° Bx). One brix is 1 gram of sucrose in 100 grams of solution and represents the strength (% w/w) of the solution as a percentage by weight (strictly, by mass). In one embodiment, a sweetener composition contains rebaudioside I in an amount effective to provide an amount equivalent to from about 0.50 to 14 brix when present in a sweetened composition, such as, for example, from about 5 to about 11 brix, from about 4 to about 7 brix, or about 5 brix. In yet another embodiment, the composition comprising rebaudioside I and at least one other sweetener are present in an amount effective to provide any one of the sweetness equivalents listed above.

In one embodiment, rebaudioside I is present in the sweetener composition in an amount effective to provide the following concentrations when the sweetener composition is added to a consumable (e.g., a beverage): from about 1ppm to about 10,000ppm, such as, for example, from about 5ppm to about 10,000ppm, from about 10ppm to about 10,000ppm, from about 15ppm to about 10,000ppm, from about 20ppm to about 10,000ppm, from about 25ppm to about 10,000ppm, from about 50ppm to about 10,000ppm, from about 100ppm to about 10,000ppm, from about 200ppm to about 10,000ppm, from about 300ppm to about 10,000ppm, from about 400ppm to about 10,000ppm, from about 500ppm to about 10,000ppm, from about 600ppm to about 10,000ppm, from about 700ppm to about 10,000ppm, from about 800 to about 10,000ppm, from about 900ppm to about 10,000ppm, from about 1,000ppm to about 10,000ppm, from about 2,000ppm to about 10,000ppm, from about 3,000ppm to about 10,000ppm, from about 4,000ppm to about 5,000 ppm. In another embodiment, rebaudioside I is present in the sweetener composition in an amount effective to provide the following concentrations when the sweetener composition is added to a consumable: from about 10ppm to about 1,000ppm, such as, for example, from about 10ppm to about 800ppm, from about 50ppm to about 600ppm or from about 200ppm to about 250 ppm. In a particular embodiment, rebaudioside I is present in the sweetener composition in an amount effective to provide a concentration of from about 300ppm to about 600ppm when the sweetener composition is added to a consumable.

In one embodiment, rebaudioside I is present in the sweetener composition in an amount effective to provide a concentration between about 400 to about 800ppm when the sweetener composition is added to a consumable, such as, for example, from about 50ppm to about 100ppm, about 100ppm to about 150ppm, about 200ppm to about 250ppm, about 250ppm to about 300ppm, about 300ppm to about 350ppm, about 350ppm to about 400ppm, about 400ppm to about 450ppm, about 450ppm to about 500ppm, about 500ppm to about 550ppm, about 550ppm to about 600ppm, about 600ppm to about 650ppm, about 650ppm to about 700ppm, about 700ppm to about 750ppm, or about 750ppm to about 800 ppm.

In an exemplary embodiment, rebaudioside I is present in the sweetener composition in an amount effective to provide a concentration between about 400 to about 800ppm when the sweetener composition is added to a beverage.

In an exemplary embodiment, rebaudioside I is present in the sweetener composition in an amount effective to provide a concentration between about 500 to about 600ppm when the sweetener composition is added to a beverage.

When the sweetener composition includes rebaudioside I in combination with one or more sweetener compounds, the amount of sweetener compound can vary. In one embodiment, the sweetener composition is present in the sweetener composition in an amount effective to provide a concentration of between about 1% and about 20%, such as between about 1% and about 5%, between about 5% and about 10%, between about 10% and about 15%, between about 15% and about 20%, or more specifically, about 1%, about 2%, about 3%, 4%, or about 5%, when the sweetener compound is added to a consumable.

In one exemplary embodiment, the present invention provides a sweetener composition comprising (I) rebaudioside I in an amount effective to provide a concentration between about 500 and about 600ppm when the sweetener composition is added to a beverage; and (ii) a compound selected from the group consisting of: rebaudioside A, B, C, D, E, M, N, O, M2, D2, glycosylated steviol glycoside, mogroside V, erythritol, psicose, stevia extract, luo han guo extract, and combinations thereof.

In one exemplary embodiment, the present invention provides a sweetener composition comprising (I) rebaudioside I in an amount effective to provide a concentration between about 500 and about 600ppm when the sweetener composition is added to a beverage; and (ii) an amount of psicose effective to provide a concentration of between about 1% and about 5% when the sweetener composition is added to a beverage.

In another embodiment, the amount present in the sweetener composition is effective to provide a concentration of about 50ppm, about 100ppm, about 150ppm, about 200ppm, about 250ppm, about 300ppm, about 350ppm, about 400ppm, about 450ppm, about 500ppm, about 550ppm, about 600ppm, about 650ppm, about 700ppm, about 750 ppm, or about 800 ppm when the sweetener composition is added to a consumable.

In an exemplary embodiment, rebaudioside I is present in the sweetener composition in an amount effective to provide a concentration of about 550ppm when the composition is added to a beverage. Optionally, the sweetener composition further comprises, for example, between about 1% and about 5%, or more particularly about 3.5% psicose.

In an exemplary embodiment, rebaudioside I is present in the sweetener composition in an amount effective to provide a concentration of about 600ppm when the composition is added to a beverage. Optionally, the sweetener composition further comprises, for example, between about 1% and about 5%, or more particularly about 3.5% psicose.

In exemplary embodiments, sweetener compositions comprising rebaudioside I exhibit a reduced sweetness linger intensity than rebaudioside M or sweetener compositions comprising it. In a particular embodiment, the isolated and purified rebaudioside I or a composition comprising rebaudioside I exhibits a sweetness linger intensity that is reduced by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% over rebaudioside M or a sweetener composition comprising it.

In some embodiments, rebaudioside I is present in the sweetener composition in an amount effective to provide a concentration of the compound above, at, or below its threshold sweetener recognition level when the sweetener composition is added to a consumable (e.g., a beverage).

Flavor enhancing compositions

In one aspect, the present invention is a flavor enhancing composition comprising rebaudioside I. In a particular embodiment, the present invention is a flavor enhancing composition comprising isolated and purified rebaudioside I.

As used herein, "flavor enhancer composition" refers to a composition that is capable of enhancing or augmenting the perception of a particular flavor in a consumable. The terms "flavor enhancing composition" or "flavor enhancer" are synonymous with the terms "flavor potentiator", "flavor amplifier", and "flavor enhancer". Generally, the flavor enhancing compositions provided herein can enhance or enhance the taste of a flavor component, i.e., any substance that provides sweetness, sourness, saltiness, aroma, bitterness, metallic taste, astringency, lingering sweet aftertaste, sweetness onset, and the like. Without being bound by any theory, the flavor enhancing composition may not provide any significant taste to the consumable to which it is added because rebaudioside I is present in the consumable at a concentration at or below its flavor recognition threshold concentration.

As used herein, "flavor recognition threshold concentration" refers to the lowest concentration of a particular flavor or aftertaste of a component (e.g., a compound) that is perceptible in a consumable. The flavor recognition threshold concentration varies for different compounds and may vary relative to the individual or specific consumable product in which the flavor is perceived.

In one embodiment, the flavor enhancing composition comprises rebaudioside I in an amount effective to provide a concentration at or below the threshold flavor recognition concentration of rebaudioside I when the flavor enhancing composition is added to a consumable.

In a particular embodiment, rebaudioside I is present in the flavor enhancing composition in an amount effective to provide a concentration below the threshold flavor recognition concentration of rebaudioside I when the flavor enhancing composition is added to a consumable.

In certain embodiments, rebaudioside I is present in the flavor enhancing composition in an amount effective to provide a concentration that is at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% or greater below a threshold flavor recognition concentration when the flavor enhancing composition is added to a consumable.

In some embodiments, rebaudioside I is present in the flavor enhancing composition in an amount that provides a concentration ranging from 1ppm to about 10,000ppm when added to a consumable (e.g., a beverage), such as, for example, from about 5ppm to about 10,000ppm, from about 10ppm to about 10,000ppm, from about 15ppm to about 10,000ppm, from about 20ppm to about 10,000ppm, from about 25ppm to about 10,000ppm, from about 50ppm to about 10,000ppm, from about 100ppm to about 10,000ppm, from about 200ppm to about 10,000ppm, from about 300ppm to about 10,000ppm, from about 400ppm to about 10,000ppm, from about 500ppm to about 10,000ppm, from about 600ppm to about 10,000ppm, from about 700ppm to about 10,000ppm, from about 800 to about 10,000ppm, from about 900ppm to about 10,000ppm, from about 1,000 to about 10,000ppm, from about 2,000 ppm to about 10,000ppm, from about 10,000ppm to about 10,000ppm, from about 10,000 ppm. In some embodiments, rebaudioside I is present in the flavor enhancing composition in an amount that provides a concentration ranging from 1ppm to about 1,000ppm when added to a consumable (e.g., a beverage), such as, for example, from about 5ppm to about 1,000ppm, from about 10ppm to about 1,000ppm, from about 20ppm to about 1,000ppm, from about 30ppm to about 1,000ppm, from about 40ppm to about 1,000ppm, from about 50ppm to about 1000ppm, from about 100ppm to about 1,000ppm, from about 200ppm to about 1,000ppm, from about 300ppm to about 1,000ppm, from about 400ppm to about 1,000ppm, and from about 500ppm to about 1,000 ppm.

One of ordinary skill in the art would be able to select the concentration of rebaudioside I in the flavor enhancing composition such that it can impart an enhanced flavor to a consumable comprising at least one flavor ingredient. For example, a skilled artisan can select the concentration of rebaudioside I in the flavor enhancing composition such that the flavor enhancing composition and/or the rebaudioside I does not impart any perceptible flavor to a consumable when the flavor enhancing composition is added to a consumable.

In one embodiment, the addition of the flavor enhancing composition increases the detected flavor of at least one flavoring ingredient in the consumable as compared to the detected flavor of the same ingredient in the consumable in the absence of the flavor enhancer.

Suitable flavoring ingredients include, but are not limited to, vanillin, vanilla extract, mango extract, cinnamon, citrus, coconut, ginger, cajeput alcohol, almond, menthol (including menthol without mint), grape skin extract, and grape seed extract. "flavoring agent" and "flavoring ingredient" are synonymous and may include natural or synthetic substances or combinations thereof. Flavoring agents also include any other substance that imparts a flavor and may include natural or non-natural (synthetic) substances that are safe for humans or animals when used in a generally accepted range. Non-limiting examples of specific flavoring agents include

Natural flavoring sweetness enhancer K14323(

Symrise of damstadt (Darmstadt, Germany), sweeteners 161453 and 164126^TMNatural seasoning covers (Symrise)^TMHall Minden (Germany), Natural Advantage^TM Bitter taste blockers 1, 2, 9 and 10(Natural Advantage)^TMFricheld, New Jersey, U.S. A., N.J.), and Surramask^TM(Creative Research Management, Stockton, California, U.S. A.)) U.S.A. was manufactured.

In another embodiment, the rebaudioside I-containing flavor enhancer composition when added to the consumable enhances flavor (either alone or in combination). Alternatively, rebaudioside I can be added directly to the consumable, i.e., not provided in a composition, to enhance flavor. In this embodiment, rebaudioside I is a flavor enhancer and is added to the consumable at a concentration at or below its threshold flavor recognition concentration.

In a particular embodiment, the flavor enhancing composition is a sweet taste enhancing composition. As used herein, "sweet taste enhancing composition" refers to a composition that is capable of enhancing or enhancing the perception of sweetness of a consumable (e.g., a beverage). The term "sweetness enhancer" is synonymous with the terms "sweetness tract enhancer", "sweetness enhancer" and "sweetness enhancer".

As used herein, a "sweet taste recognition threshold concentration" is the lowest known concentration of a sweet compound that is perceivable by a human taste. In general, the sweetness enhancing compositions of the present invention can enhance or enhance the sweetness tract of a consumable without providing any significant sweetness per se, because the rebaudioside I concentration in the sweetness enhancing composition is at or below its sweetness recognition threshold concentration, in the sweetness enhancing composition, in the consumable after addition of the sweetness enhancing composition, or both. The sweetness recognition threshold concentration is specific to a particular compound and may vary based on temperature, substrate, ingredient, and/or flavor system.

In one embodiment, the sweetness enhancing composition comprises rebaudioside I in an amount effective to provide a concentration at or below the threshold sweetness recognition concentration of rebaudioside I when the sweetness enhancing composition is added to a consumable.

In a particular embodiment, the sweetness enhancing composition comprises rebaudioside I in an amount effective to provide a concentration below the threshold sweetness recognition concentration of rebaudioside I when the sweetness enhancing composition is added to a consumable.

In certain embodiments, rebaudioside I is present in the sweetness enhancing composition in an amount effective to provide a concentration that is at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% or more below the threshold sweetness recognition concentration when the sweetness enhancing composition is added to a consumable.

In some embodiments, rebaudioside I is present in the sweetness enhancing composition in an amount that provides a concentration from about 1ppm to about 10,000ppm when added to a consumable (e.g., a beverage), such as, for example, from about 5ppm to about 10,000ppm, from about 10ppm to about 10,000ppm, from about 15ppm to about 10,000ppm, from about 20ppm to about 10,000ppm, from about 25ppm to about 10,000ppm, from about 50ppm to about 10,000ppm, from about 100ppm to about 10,000ppm, from about 200ppm to about 10,000ppm, from about 300ppm to about 10,000ppm, from about 400ppm to about 10,000ppm, from about 500ppm to about 10,000ppm, from about 600ppm to about 10,000ppm, from about 700ppm to about 10,000ppm, from about 800 to about 10,000ppm, from about 900ppm to about 10,000ppm, from about 1,000 to about 10,000ppm, from about 2 ppm to about 10,000ppm, from about 10,000ppm to about 10,000ppm, from about 10,000 ppm. In some embodiments, rebaudioside I is present in the sweetness enhancing composition in an amount that provides a concentration ranging from 1ppm to about 1,000ppm when added to a consumable (e.g., a beverage), such as, for example, from about 5ppm to about 1,000ppm, from about 10ppm to about 1,000ppm, from about 20ppm to about 1,000ppm, from about 30ppm to about 1,000ppm, from about 40ppm to about 1,000ppm, from about 50ppm to about 1000ppm, from about 100ppm to about 1,000ppm, from about 200ppm to about 1,000ppm, from about 300ppm to about 1,000ppm, from about 400ppm to about 1,000ppm, and from about 500ppm to about 1,000 ppm.

Alternatively, rebaudioside I may be added directly to the consumable, i.e., not provided in the form of a composition, to enhance sweetness. In this embodiment, rebaudioside I is a sweetness enhancer and is added to the consumable at a concentration at or below its sweetness recognition threshold concentration.

The sweetness of a given composition is typically measured with reference to a sucrose solution. See generally "Systematic studies of sweetener Concentration-Response Relationships" (a Systematic Study of Concentration-Response Relationships of sweentees) ", g.e. dub (g.e. DuBois), d.e. walters (d.e. walters), s.s. schfmann (s.s.schiffman), z.s. walick (z.s.walwick), b.j. bott (b.j.booth), s.d. picory (s.d. Pecore), k. gibis (k.gibes), b.t. karl (b.t.carr), and l.m. brunett (l.m.brands), in the sweetener: discovery, Molecular Design and chemosensing (Sweenters: Discovery, Molecular Design and Chemoreception), D.E. Wolters, F.T. Orthoefer and G.E. Dubraw editions, American Chemical Society, Washington (Washington), DC (1991), page 261-.

It is contemplated that the sweetness enhancing composition can include one or more sweetness enhancers other than rebaudioside I. In one embodiment, the sweetness enhancing composition may comprise an additional sweetness enhancer. In other embodiments, the sweetness enhancing composition may include two or more additional sweetness enhancers. In embodiments utilizing two or more sweetness enhancers, each sweetness enhancer should be present below its respective sweetness recognition threshold concentration.

Suitable sweetness enhancers include, but are not limited to, the group consisting of: 2-hydroxybenzoic acid, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 2, 4-dihydroxybenzoic acid, 3, 4-dihydroxybenzoic acid, 2, 5-dihydroxybenzoic acid, 2, 6-dihydroxybenzoic acid, 2,3, 4-trihydroxybenzoic acid, 2,4, 6-trihydroxybenzoic acid, 3-aminobenzoic acid, 4-aminobenzoic acid, FEMA GRAS enhancer 4469, FEMA GRAS enhancer 4701, FEMA GRAS enhancer 4720, FEMA GRAS enhancer 4774, FEMA GRAS enhancer 4708, FEMA GRAS enhancer 4728, FEMA GRAS enhancer 4601, and combinations thereof.

In one embodiment, the addition of the one or more sweetness enhancers increases the detected sucrose equivalence of the at least one sweetener in a consumable as compared to the sucrose equivalence in the same consumable in the absence of the sweetness enhancer.

More specifically, the use of rebaudioside I and optionally one or more other sweetness enhancers (alone or in a composition) in a consumable (e.g., a beverage) provides a detected sucrose equivalent that is at least about 0.5% greater than the sucrose equivalent of a corresponding consumable (e.g., a beverage) in the absence of rebaudioside I and optionally one or more other sweetness enhancers. For example, the measured sucrose equivalent of a consumable (e.g., a beverage) comprising rebaudioside I and optionally one or more other sweetness enhancers, alone or in a composition, can be at least about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.5%, about 5.0%, or about 5.5% or more greater than the sucrose equivalent of a corresponding consumable in the absence of rebaudioside I and optionally one or more other sweetness enhancers.

Suitable sweeteners include, but are not limited to: sucrose, glyceraldehyde, dihydroxyacetone, erythrose, threose, erythrulose, arabinose, lyxose, ribose, xylose, ribulose, xylulose, allose, altrose, galactose, glucose, gulose, idose, mannose, talose, fructose, psicose, sorbose, tagatose, mannoheptulose, sedoheptulose, octulose, fucose, rhamnose, arabinose, turanose, salivary sugar, rebaudioside a, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside N, rebaudioside O, dulcoside a, dulcoside B, rubusoside, stevia, stevioside, mogroside IV, mogroside V, luo han guo, siamenoside, monatin and salts thereof (monatin SS, s, D, RR, RS, SR), curculin (curculin), glycyrrhizic acid and its salt, thaumatin, monellin (monellin), mabinlin (mabinlin), brazzein (brazzein), southeast Doxin (hernandulcin), phyllodulcin, phloridzin, trilobatin, anabasine (baiyunoside), Osladin (osladin), polyvidone (polypodoside) A, pterocaryoside (pterocaryoside) A, pterocaryoside B, mucronoside, fimisoside I, glycyrrhizin I, abrin triterpenoid A, steviolbioside and cyclocarioside I, sugar alcohols such as erythritol, sucralose, acesulfame potassium, acesulfame acid and its salts, aspartame, alitame, saccharin and its salts, neohesperidin dihydrochalcone, cyclamate, cyclamic acid and its salts, neotame, saccharin (an), Glycosylated Steviol Glycosides (GSG) and combinations thereof.

In one embodiment, the sweetener is a caloric sweetener or a mixture of caloric sweeteners. In another embodiment, the caloric sweetener is selected from the group consisting of sucrose, fructose, glucose, high fructose corn/starch syrup, beet sugar, sucrose, and combinations thereof.

In another embodiment, the sweetener is a rare sugar selected from the group consisting of D-psicose, L-ribose, D-tagatose, L-glucose, L-trehalose, L-arabinose, turanose, and combinations thereof.

In yet another embodiment, the sweetener is a non-caloric sweetener or a mixture of non-caloric sweeteners. In one example, the non-caloric sweetener is a natural high-potency sweetener. As used herein, the phrase "natural high-potency sweetener" refers to any composition that is not naturally found in nature and characteristically has a sweetness potency greater than sucrose, fructose, or glucose, yet has a smaller calorie. Natural high-potency sweeteners may be provided as a pure compound or alternatively as part of an extract.

In yet another example, the non-caloric sweetener is a synthetic high-potency sweetener. As used herein, the phrase "synthetic sweetener" refers to any composition not naturally found in nature and characteristically having a sweetness potency greater than sucrose, fructose, or glucose, yet having a smaller calorie.

In a particular embodiment, the consumable product is a beverage. The beverage comprises rebaudioside I and at least one sweetener, wherein rebaudioside I is present at a concentration at or below its sweetness recognition threshold. Rebaudioside I and at least one sweetener may each be provided separately, or in the form of a sweetness enhancing composition. In a particular embodiment, the detected sucrose equivalent is increased from, e.g., about 0.2% to about 5.0%, e.g., about 1%, about 2%, about 3%, about 4%, or about 5%.

The sweetener can be any of the natural or synthetic sweeteners provided herein. In one particular embodiment, the sweetener is a calorie-providing carbohydrate sweetener. Thus, incorporation of the sweetness enhancer thereby reduces the amount of calorie-providing carbohydrate sweetener that must be used in a given consumable, thereby allowing for the preparation of a reduced calorie consumable.

The composition can be tailored to provide a desired caloric content. For example, the compositions may be "calorie-rich" such that they impart a desired sweetness when added to a consumable product (e.g., like a beverage) and have about 120 calories per 8 ounce serving. Alternatively, the compositions may be "median caloric" such that they impart the desired sweetness when added to a consumable (e.g., like a beverage) and have less than about 60 calories per 8 ounce serving. In other embodiments, the compositions may be "diet" such that they impart a desired sweetness when added to a consumable product (e.g., like a beverage) and have less than 40 calories per 8 ounce serving. In still other embodiments, the compositions may be "zero calorie" such that they impart a desired sweetness when added to a consumable (e.g., like a beverage) and have less than 5 calories per 8 ounce serving.

Additive agent

Compositions, such as sweetener compositions and flavor enhancing compositions, can comprise one or more additives in addition to rebaudioside I, as described in detail below. In some embodiments, the composition comprises additives including, but not limited to, carbohydrates, polyols, amino acids and their corresponding salts, polyamino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic salts (including organic acid salts and organic base salts), inorganic salts, bitter compounds, flavoring and flavoring ingredients, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, weighting agents (weighing agents), gums (gum), antioxidants, colorants, flavonoids, alcohols, polymers, and combinations thereof. In some embodiments, the additives are used to modify the temporal profile and flavor profile of the sweetener to provide a sweetener composition with a taste similar to sucrose.

In one embodiment, the composition further comprises one or more polyols. As used herein, the term "polyol" refers to a molecule containing more than one hydroxyl group. The polyol may be a diol, triol or tetraol containing 2, 3 and 4 hydroxyl groups respectively. The polyols may also contain more than 4 hydroxyl groups, for example, pentahydric, hexahydric, heptahydric alcohols, and the like, containing 5, 6, or 7 hydroxyl groups, respectively. In addition, a polyol can also be a sugar alcohol, a polyhydroxy alcohol or a polyol (polyalchol) in reduced form of a carbohydrate, wherein the carbonyl groups (aldehyde or ketone, reducing sugar) have been reduced to primary or secondary hydroxyl groups.

Non-limiting examples of polyols in some embodiments include erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, isomalt, propylene glycol, glycerol (glycerol), threitol, galactitol, palatinose, reduced isomaltooligosaccharides, reduced xylooligosaccharides, reduced gentiooligosaccharides, reduced maltose syrups, reduced glucose syrups, and sugar alcohols or any other carbohydrate capable of being reduced, which does not adversely affect the taste of the composition.

In certain embodiments, the polyol, when present in a consumable (such as, for example, a beverage), is present in the composition in an amount effective to provide a concentration from about 100ppm to about 250,000 ppm. In other embodiments, the polyol, when present in a consumable (such as, for example, a beverage), is present in the composition in an amount effective to provide a concentration from about 400ppm to about 80,000ppm, such as, for example, from about 5,000ppm to about 40,000 ppm.

In other embodiments, rebaudioside I and polyol are present in the composition in a weight ratio of from about 1:1 to about 1:800, such as, for example, from about 1:4 to about 1:800, from about 1:20 to about 1:600, from about 1:50 to about 1:300, or from about 1:75 to about 1: 150.

Suitable amino acid additives include, but are not limited to, aspartic acid, arginine, glycine, glutamic acid, proline, threonine, theanine, cysteine, cystine, alanine, valine, tyrosine, leucine, arabinose, trans-4-hydroxyproline, isoleucine, asparagine, serine, lysine, histidine, ornithine, methionine, carnitine, aminobutyric acid (α, β, and/or δ -isomers), glutamine, hydroxyproline, taurine, norvaline, sarcosine, and salt forms thereof such as sodium or potassium salts or acid salts. These amino acid additives may also be in the D-configuration or L-configuration and in the mono-, di-or tri-form of the same or different amino acids. In addition, these amino acids can, if appropriate, be the α -, β -, γ -and/or δ -isomers. In some embodiments, combinations of the above amino acids and their corresponding salts (e.g., their sodium, potassium, calcium, magnesium or other alkali or alkaline earth metal salts, or acid salts) are also suitable. These amino acids may be natural or synthetic. These amino acids may also be modified. A modified amino acid refers to any amino acid in which at least one atom has been added, removed, substituted, or a combination thereof (e.g., an N-alkyl amino acid, an N-acyl amino acid, or an N-methyl amino acid). Non-limiting examples of modified amino acids include amino acid derivatives such as trimethylglycine, N-methyl-glycine, and N-methyl-alanine. As used herein, modified amino acids encompass both modified and unmodified amino acids. As used herein, amino acids also encompass both peptides and polypeptides (e.g., dipeptides, tripeptides, tetrapeptides, and pentapeptides), such as glutathione and L-alanyl-L-glutamine. Suitable polyamino acid additives include poly-L-aspartic acid, poly-L-lysine (e.g., poly-L-alpha-lysine or poly-L-epsilon-lysine), poly-L-ornithine (e.g., poly-L-alpha-ornithine or poly-L-epsilon-ornithine), poly-L-arginine, other polymeric forms of amino acids, and salt forms thereof (e.g., calcium, potassium, sodium, or magnesium salts, such as L-glutamic acid monosodium salt). The polyamino acid additive may also be in the D-configuration or the L-configuration. In addition, the polyamino acids may, if appropriate, be the α -, β -, γ -, δ -and ε -isomers. In some embodiments, combinations of the above polyamino acids and their corresponding salts (e.g., their sodium, potassium, calcium, magnesium or other alkali or alkaline earth metal salts or acid salts) are also suitable. The polyamino acids described herein may also include copolymers of different amino acids. These polyamino acids may be natural or synthetic. The polyamino acid may also be modified such that at least one atom is added, removed, substituted, or a combination thereof (e.g., an N-alkyl polyamino acid or an N-acyl polyamino acid). As used herein, polyamino acids encompass both modified and unmodified polyamino acids. For example, modified polyamino acids include, but are not limited to, polyamino acids having different Molecular Weights (MW), such as poly-L-a-lysine having a MW of 1,500, a MW of 6,000, a MW of 25,200, a MW of 63,000, a MW of 83,000, or a MW of 300,000.

In particular embodiments, the amino acid, when present in a consumable (such as, for example, a beverage), is present in the composition in an amount effective to provide a concentration of from about 10ppm to about 50,000 ppm. In another embodiment, the amino acid, when present in a consumable product, is present in the composition in an amount effective to provide a concentration of from about 1,000ppm to about 10,000ppm, such as, for example, from about 2,500ppm to about 5,000ppm or from about 250ppm to about 7,500 ppm.

Suitable sugar acid additives include, but are not limited to, aldonic acids, uronic acids, aldaric acids, alginic acids, gluconic acids, glucuronic acids, glucaric acids, galactaric acids, galacturonic acids, and salts thereof (e.g., sodium, potassium, calcium, magnesium, or other physiologically acceptable salts), and combinations thereof.

Suitable nucleotide additives include, but are not limited to, inosine monophosphate ("IMP"), guanosine monophosphate ("GMP"), adenosine monophosphate ("AMP"), Cytosine Monophosphate (CMP), Uracil Monophosphate (UMP), inosine diphosphate, guanosine diphosphate, adenosine diphosphate, cytosine diphosphate, uracil diphosphate, inosine triphosphate, guanosine triphosphate, adenosine triphosphate, cytosine triphosphate, uracil triphosphate, alkali metal or alkaline earth metal salts thereof, and combinations thereof. The nucleotides described herein can also include nucleotide-related additives such as nucleosides or nucleic acid bases (e.g., guanine, cytosine, adenine, thymine, uracil).

When present in a consumable (such as, for example, a beverage), the nucleotide is present in the composition in an amount effective to provide a concentration of from about 5ppm to about 1,000 ppm.

Suitable organic acid additives include any compound comprising a-COOH moiety, such as, for example, C2-C30 carboxylic acids, substituted hydroxy C2-C30 carboxylic acids, butyric (ethyl) acid, substituted butyric (ethyl) acid, benzoic acid, substituted benzoic acid (e.g., 2, 4-dihydroxybenzoic acid), substituted cinnamic acids, hydroxy acids, substituted hydroxybenzoic acids, anisic acid substituted cyclohexylcarboxylic acids, tannic acid, aconitic acid, lactic acid, tartaric acid, citric acid, isocitric acid, gluconic acid, glucoheptonic acid, adipic acid, hydroxycitric acid, malic acid, fruit tartaric acid (fruitaric acid) (a blend of malic acid, fumaric acid, maleic acid, succinic acid, chlorogenic acid, salicylic acid, creatine, caffeic acid, bile acid, acetic acid, ascorbic acid, alginic acid, isoascorbic acid, polyglutamic acid, succinic acid, fumaric acid, succinic acid, glucono delta lactone, and alkali metal salt or alkaline earth metal salt derivatives thereof. In addition, the organic acid additive may also be in the D-configuration or the L-configuration.

Suitable organic acid additive salts include, but are not limited to, sodium, calcium, potassium, and magnesium salts of all organic acids, such as citric acid, malate, tartrate, fumarate, lactate (e.g., sodium lactate), alginate (e.g., sodium alginate), ascorbate (e.g., sodium ascorbate), benzoate (e.g., sodium or potassium benzoate), sorbate, and adipate. Examples of the organic acid additive may optionally be substituted with at least one group selected from: hydrogen, alkyl, alkenyl, alkynyl, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino, amido, carboxyl derivative, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, thiol, imine, sulfonyl, thiolate, sulfinyl, sulfamoyl, carboxyalkoxy, carbonamide (carboxamido), phosphonyl, phosphinyl, phosphoryl, phosphino, thioester, thioether, anhydride, oximino, hydrazino, carbamoyl, phosphorus, or phosphonate. In particular embodiments, the organic acid additive, when present in a consumable (such as, for example, a beverage), is present in the composition in an amount effective to provide a concentration of from about 10ppm to about 5,000 ppm.

Suitable inorganic acid additives include, but are not limited to, phosphoric acid, phosphorous acid, polyphosphoric acid, hydrochloric acid, sulfuric acid, carbonic acid, sodium dihydrogen phosphate, and alkali or alkaline earth metal salts thereof (e.g., inositol hexaphosphate Mg/Ca).

When present in a consumable (such as, for example, a beverage), the mineral acid additive is present in the composition in an amount effective to provide a concentration of from about 25ppm to about 25,000 ppm.

Suitable bitter compound additives include, but are not limited to, caffeine, quinine, urea, bitter orange oil, naringin, quassia, and salts thereof.

When present in a consumable (such as, for example, a beverage), the bitter compound is present in the composition in an amount effective to provide a concentration of from about 25ppm to about 25,000 ppm.

Suitable flavoring agents and ingredients include, but are not limited to, vanillin, vanilla extract, mango extract, cinnamon, citrus, coconut, ginger, melaleuca (viridiflorol), almond kernel, menthol (including menthol without mint), grape skin extract, and grape seed extract. "flavoring agent" and "flavoring ingredient" are synonymous and may include natural or synthetic substances or combinations thereof. Flavoring agents also include any other substance that imparts a flavor and may include natural or non-natural (synthetic) substances that are safe for humans or animals when used in a generally accepted range. Non-limiting examples of specific flavoring agents include

Natural flavoring sweetness enhancer K14323(

Symrise of damstadt (Darmstadt, Germany), sweeteners 161453 and 164126^TMNatural seasoning covers (Symrise)^TMHall Minden (Germany), Natural Advantage^TM Bitter taste blockers 1, 2, 9 and 10(Natural Advantage)^TMFricheld, New Jersey, U.S. A., N.J.), and Surramask^TM(Creative Research Management, Stockton, Ca, Calif., USA)lifornia, U.S.A.))。

When present in a consumable product (such as, for example, a beverage), the flavoring agent is present in the composition in an amount effective to provide a concentration of from about 0.1ppm to about 4,000 ppm.

Suitable polymeric additives include, but are not limited to, chitin, pectin, pectic acid, polyuronic acid, polygalacturonic acid, starch, food hydrocolloids or crude extracts thereof (e.g., acacia senegal (Fibergum)^TM) Gum arabic, carageenan), poly-L-lysine (e.g., poly-L- α -lysine or poly-L-e-lysine), poly-L-ornithine (e.g., poly-L- α -ornithine or poly-L-e-ornithine), polypropylene glycol, polyethylene glycol, poly (ethylene glycol methyl ether), polyarginine, polyaspartic acid, polyglutamic acid, polyethyleneimine, alginic acid, sodium alginate, propylene glycol alginate, and sodium polyethylene glycol alginate, sodium hexametaphosphate and salts thereof, and other cationic and anionic polymers.

When present in a consumable product (such as, for example, a beverage), the polymer is present in the composition in an amount effective to provide a concentration of from about 30ppm to about 2,000 ppm.

Suitable protein or protein hydrolysate additives include, but are not limited to, Bovine Serum Albumin (BSA), whey protein (including fractions or concentrates thereof, e.g., 90% instant whey protein isolate, 34% whey protein, 50% hydrolyzed whey protein, and 80% whey protein concentrate), soluble rice protein, soy protein, protein isolates, protein hydrolysates, reaction products of protein hydrolysates, glycoproteins, and/or proteoglycans containing amino acids (e.g., glycine, alanine, serine, threonine, asparagine, glutamine, arginine, valine, isoleucine, leucine, norvaline, methionine, proline, tyrosine, hydroxyproline, and the like), collagen (e.g., gelatin), partially hydrolyzed collagen (e.g., hydrolyzed fish collagen), and collagen hydrolysates (e.g., porcine collagen hydrolysate).

When present in a consumable (such as, for example, a beverage), the protein hydrolysate is present in the composition in an amount effective to provide a concentration of from about 200ppm to about 50,000 ppm.

Suitable surfactant additives include, but are not limited to, polysorbates (e.g., polyoxyethylene sorbitan monooleate (polysorbate 80), polysorbate 20, polysorbate 60), sodium dodecylbenzenesulfonate, dioctyl or dioctyl sodium sulfosuccinate, sodium lauryl sulfate, cetylpyridinium chloride (cetylpyridinium chloride), cetyltrimethylammonium bromide, sodium cholate, carbamoyl, choline chloride, sodium glycocholate, sodium taurodeoxycholate, lauroyl arginine ester, sodium stearoyl lactylate, sodium taurocholate, lecithin, sucrose oleate, sucrose stearate, sucrose palmitate, sucrose laurate, and other emulsifiers and the like.

When present in a consumable product (such as, for example, a beverage), the surfactant additive is present in the composition in an amount effective to provide a concentration of from about 30ppm to about 2,000 ppm.

Suitable flavonoid additives are classified as flavonols, flavones, flavanones, flavan-3-ols, isoflavones or anthocyanidins. Non-limiting examples of flavonoid additives include, but are not limited to, catechins (e.g., green tea extracts, such as Polyphenon @) ^TM60、Polyphenon ^TM30 and Polyphenon^TM25 (Mitsui Norin co., ltd., Japan)), polyphenol, rutin (e.g., enzyme-modified rutin Sanmelin)^TMAO (San-fi Gen f.f.i., inc., Osaka, Japan), neohesperidin, naringin, neohesperidin dihydrochalcone, and the like.

When present in a consumable (such as, for example, a beverage), the flavonoid additive is present in the composition in an amount effective to provide a concentration from about 0.1ppm to about 1,000 ppm.

Suitable alcohol additives include, but are not limited to, ethanol. In particular embodiments, the alcohol additive, when present in a consumable (such as, for example, a beverage), is present in the composition in an amount effective to provide a concentration of from about 625ppm to about 10,000 ppm.

Suitable astringent compounds include, but are not limited to, tannic acid, europium chloride (EuCl)₃) Gadolinium chloride (GdCl)₃) Terbium chloride (TbCl)₃) Alum, tannic acid, and polyphenols (e.g., tea polyphenols). When present in a consumable (such as, for example, a beverage), the astringency additive is present in the composition in an amount effective to provide a concentration of from about 10ppm to about 5,000 ppm.

Functional ingredients

The compositions provided herein may also contain one or more functional ingredients that provide an actual or perceived health benefit to the composition. Functional ingredients include, but are not limited to, saponins, antioxidants, dietary fiber sources, fatty acids, vitamins, glucosamine, minerals, preservatives, hydration agents, probiotics, prebiotics, weight management agents (weight management agents), osteoporosis management agents (osteoporosis management agents), phytoestrogens, long chain primary aliphatic saturated alcohols, phytosterols, and combinations thereof.

Saponin

In certain embodiments, the functional ingredient is at least one saponin. As used herein, the at least one saponin may include a single saponin or multiple saponins as a functional ingredient of the compositions provided herein. Generally, according to particular embodiments of the present invention, the at least one saponin is present in the composition in an amount sufficient to promote health and wellness.

Saponins are glycosidic natural plant products comprising an aglycone ring structure and one or more sugar moieties. The combination of the non-polar aglycone and the water-soluble sugar moiety imparts saponin surfactant properties which allow them to form a foam when shaken in an aqueous solution.

These saponins are grouped together based on several common characteristics. In particular, saponins are surfactants that exhibit hemolytic activity and form complexes with cholesterol. Although saponins share these properties, they are structurally different. The type of aglycone ring structure that forms a ring structure in saponin may vary significantly. Non-limiting examples of these types of aglycone ring structures in saponins useful in particular embodiments of the present invention include steroids, triterpenes and steroidal alkaloids. Non-limiting examples of specific aglycone ring structures useful in embodiments of the present invention include soyasapogenol a, soyasapogenol B and soyasapogenol E. The number and type of sugar moieties attached to the aglycone ring structure may also vary widely. Non-limiting examples of sugar moieties useful in particular embodiments of the present invention include glucose, galactose, glucuronic acid, xylose, rhamnose, and methyl pentose moieties. Non-limiting examples of specific saponins for use in particular embodiments of the present invention include group a acetylsaponins, group B acetylsaponins, and group E acetylsaponins.

Saponins can be found in a wide variety of plants and plant products, and are particularly prevalent in plant bark and bark, where they form a waxy protective coating. Several common sources of saponins include soy, soapwort (Saponaria, the root of which has historically been used as a soap), and alfalfa, aloe, asparagus, grapes, chickpeas, yucca, and various other legumes and weeds, with a saponin content of about 5% by dry weight. Saponins may be obtained from these sources by using extraction techniques well known to those of ordinary skill in the art. A description of conventional extraction techniques can be found in U.S. patent application No. 2005/0123662, the disclosure of which is expressly incorporated by reference.

Antioxidant agent

In certain embodiments, the functional ingredient is at least one antioxidant. As used herein, the at least one antioxidant can include a single antioxidant or multiple antioxidants as a functional ingredient of the compositions provided herein. Generally, according to particular embodiments of the present invention, the at least one antioxidant is present in the composition in an amount sufficient to promote health and wellness.

As used herein, "antioxidant" refers to any substance that inhibits, or reduces oxidative damage to cells and biomolecules. Without being bound by theory, it is believed that antioxidants prevent, inhibit, or reduce oxidative damage to cells or biomolecules by stabilizing free radicals (before they can cause harmful reactions). In this way, antioxidants may prevent or delay the onset of certain degenerative diseases.

Examples of suitable antioxidants for use in embodiments of the present invention include, but are not limited to, vitamins, vitamin cofactors, minerals, hormones, carotenoids, carotenoid terpenes, non-carotenoid terpenes, flavonoids, flavonoid polyphenols (e.g., bioflavonoids), flavonols, flavonoids, phenols, polyphenols, phenolic esters, polyphenolic esters, non-flavonoid phenols, isothiocyanates, and combinations thereof. In some embodiments, the antioxidant is vitamin a, vitamin C, vitamin E, ubiquinone, the minerals selenium, manganese, melatonin, alpha-carotene, beta-carotene, lycopene, lutein, zeaxanthin (zeaxanthin), cryptoxanthin (cryptoxanthin), resveratrol (reservatol), eugenol, quercetin, catechin, gossypol, hesperetin, curcumin, ferulic acid, thymol, hydroxytyrosol, turmeric, thyme, olive oil, lipoic acid, glutathione (glutathione), glutamine (vitamine), oxalic acid, a tocopherol derivative, Butylated Hydroxyanisole (BHT), Butylated Hydroxytoluene (BHT), ethylenediaminetetraacetic acid (EDTA), tert-butylhydroquinone, acetic acid, pectin, tocotrienols, tocopherols, coenzyme Q10, zeaxanthin, astaxanthin, canthaxanthin (canthaxanthin), saponin, vitamin a, vitamin C, vitamin E, ubiquinone, resveratrol (lutein), vitamin d, and other vitamins, such as vitamins, for example, vitamin a pharmaceutical compositions containing the antioxidant, Limonin, kaempferol (kaempfedrol), myricetin, isorhamnetin, proanthocyanidin, quercetin, rutin, luteolin, apigenin, tangeretin (tangeritin), hesperetin, naringenin, eriodictyol (erythrotyol), flavan-3-ol (e.g., anthocyanidin), gallocatechin, epicatechin and gallate forms thereof, epigallocatechin and gallate forms thereof (ECGC), theaflavin and gallate forms thereof, thearubigin, isoflavone phytoestrogens, genistein, daidzein, glycitein, anthocyanins (anythocyanin), cyanides (cyaniding), delphinidin, malvidin, methylcyanin, petunidin, ellagic acid, gallic acid, salicylic acid, rosmarinic acid, cinnamic acid and derivatives thereof (e.g., ferulic acid), chlorogenic acid, chrysanthemic acid (chicoric acid), Galla chinensis tannin, ellagitannins, anthoxanthins, beta-anthocyanins and other plant pigments, silymarin, citric acid, lignans, anti-nutrients (anti-nutritional), bilirubin, uric acid, R-alpha-lipoic acid, N-acetylcysteine, nobiletin (embilicin), apple extract, apple peel extract (apple polyphenol), Rooibos extract (rooibos extract), Rooibos extract (green), Crataegus pinnatifida extract, Rubi fructus extract, Green Coffee Antioxidant (GCA), Prunus davidiana extract 20%, grape seed extract (Vinoseed), cacao bean extract, cranberry extract, mangosteen fruit shell extract, cranberry extract, pomegranate peel extract, pomegranate seed extract, hawthorn berry extract, pomegranate (pomegranate), pomegranate fruit extract, and/extract, pomegranate fruit extract, and/extract, and its, Cinnamon bark extract, grape skin extract, bilberry extract, pine bark extract, pycnogenol, elderberry extract, mulberry root extract, wolfberry (gogi) extract, blackberry extract, blueberry leaf extract, raspberry extract, turmeric extract, citrus bioflavonoids, blackcurrant, ginger, acai berry powder, green coffee bean extract, green tea extract, and phytic acid, or a combination thereof. In alternative embodiments, the antioxidant is a synthetic antioxidant, such as, for example, butylated hydroxytoluene or butylated hydroxyanisole. Other sources of suitable antioxidants for use in embodiments of the present invention include, but are not limited to, fruits, vegetables, tea, cocoa, chocolate, spices, herbs, rice, organ meats from livestock, yeast, whole grains (cereal grains), or cereal grains (cereal grains).

Specific antioxidants belong to the group of phytonutrients called polyphenols (also called "polyphenols"), which are a group of chemical substances visible in plants, characterized by the presence of more than one phenol group per molecule. Various health benefits may stem from polyphenols including, for example, prevention of cancer, heart disease, and chronic inflammatory diseases, as well as increased mental and physical strength. Suitable polyphenols for use in embodiments of the present invention include catechins, proanthocyanidins, procyanidins, anthocyanidins, quercetin, rutin, resveratrol, isoflavones, curcumin, punicalagin, ellagitannins, hesperidins, naringin, citrus flavonoids, chlorogenic acid, other similar materials, and combinations thereof.

In particular embodiments, the antioxidant is a catechin, such as, for example, epigallocatechin gallate (EGCG). Suitable sources of catechins for use in embodiments of the present invention include, but are not limited to, green tea, white tea, black tea, oolong tea, chocolate, cocoa, red wine, grape seed, red grape skin, purple grape skin, red grape juice, purple grape juice, berries, pycnogenol, and red apple skin.

In some embodiments, the antioxidant is selected from proanthocyanidins, procyanidins, or combinations thereof. Suitable sources of proanthocyanidins and procyanidins for use in embodiments of the invention include, but are not limited to, red grapes, purple grapes, cocoa, chocolate, grape seeds, red wine, cocoa beans, cranberry, apple peel, plum, blueberry, blackcurrant, chokeberry (choke berry), green tea, sorghum, cinnamon, barley, red kidney beans, black and white speckles, hops, almonds, hazelnuts, pecans, pistachio fruits, pycnogenol, and colorberries.

In particular embodiments, the antioxidant is an anthocyanin. Suitable sources of anthocyanins for embodiments of the present invention include, but are not limited to, red berries, blueberries, bilberries, cranberries, raspberries, cherries, pomegranates, strawberries, elderberries, chokeberries, red grape skin, purple grape skin, grape seeds, red wine, black currants, red currants, cocoa, plums, apple skins, peaches, red pears, red cabbage, red onions, red oranges, and blackberries.

In some embodiments, the antioxidant is selected from quercetin, rutin, or a combination thereof. Suitable sources of quercetin and rutin for use in embodiments of the present invention include, but are not limited to, red apple, onion, kale, vaccinium uliginosum, bilberry, chokeberry, cranberry, blackberry, blueberry, strawberry, raspberry, black currant, green tea, black tea, plum, apricot, parsley, leek, broccoli, red pepper, berry wine, and ginkgo biloba.

In some embodiments, the antioxidant is resveratrol. Suitable sources of resveratrol for use in embodiments of the invention include, but are not limited to, red grapes, peanuts, cranberries, blueberries, cranberries, mulberries, Japanese teas (Japanese Itadori tea), and red wine.

In a particular embodiment, the antioxidant is an isoflavone. Suitable sources of isoflavones for use in embodiments of the present invention include, but are not limited to, soybeans, soybean products, legumes, alfalfa sprouts (alfalfalfa spits), chickpeas, peanuts, and red clover.

In some embodiments, the antioxidant is curcumin. Suitable sources of curcumin for use in embodiments of the present invention include, but are not limited to, turmeric and mustard.

In particular embodiments, the antioxidant is selected from quercetin, ellagitannin, or a combination thereof. Suitable sources of quercetin and ellagitannins for use in embodiments of the present invention include, but are not limited to, pomegranate, raspberry, strawberry, walnut, and older red wine.

In some embodiments, the antioxidant is a citrus flavonoid, such as hesperidin or naringin. Suitable sources of citrus flavonoids such as hesperidin or naringin for use in embodiments of the present invention include, but are not limited to, orange, grapefruit, and citrus juices.

In a particular embodiment, the antioxidant is chlorogenic acid. Suitable sources of chlorogenic acid for embodiments of the present invention include, but are not limited to, raw coffee, yerba mate, red wine, grape seed, red grape skin, purple grape skin, red grape juice, purple grape juice, apple juice, cranberry, pomegranate, blueberry, strawberry, sunflower, echinacea, pycnogenol, and apple peel.

Dietary fiber

In certain embodiments, the functional ingredient is at least one source of dietary fiber. As used herein, the at least one dietary fiber source may include a single dietary fiber source or multiple dietary fiber sources as a functional ingredient of the compositions provided herein. Generally, according to particular embodiments of the present invention, the at least one source of dietary fiber is present in the composition in an amount sufficient to promote health and wellness.

A variety of polymeric carbohydrates having significantly different structures in both composition and linkage fall within the definition of dietary fiber. Such compounds are well known to those skilled in the art, and non-limiting examples thereof include non-starch polysaccharides, lignin, cellulose, methyl cellulose, hemicellulose, beta-glucan, pectin, gums, mucilage, waxes, inulin, oligosaccharides, fructooligosaccharides, cyclodextrins, chitin, and combinations thereof.

Polysaccharides are complex carbohydrates composed of monosaccharides linked by glycosidic bonds. Non-starch polysaccharides bind to beta bonds and one cannot digest them due to the lack of an enzyme that breaks the beta bonds. In contrast, digestible starch polysaccharides typically contain alpha (1-4) linkages.

Lignin is a large, highly branched and cross-linked polymer based on oxyphenyl propane units. Cellulose is a linear polymer of glucose molecules linked by a β (1-4) linkage, which mammalian amylases are unable to hydrolyze. Methylcellulose is a methyl ester of cellulose that is commonly used in food products as a thickener and emulsifier. It is commercially available (e.g., Citrecel, marketed by Kurarin Schker (GlaxoSmithKline), Celevac, marketed by Sera Pharmaceuticals (fire Pharmaceuticals)). Hemicelluloses are highly branched polymers composed mainly of glucuronic acid-and 4-O-methylglucuronoxylan. Beta glucans are mixed-bond (1-3), (1-4) beta-D-glucose polymers found primarily in cereals such as oat and barley. Pectins such as beta pectin are a group of polysaccharides consisting essentially of D-galacturonic acid, which is methoxylated to varying degrees.

Gums and mucilages represent a wide array of differently branched structures. Guar gum derived from the milled endosperm of guar seeds is a galactomannan. Guar gum is commercially available (e.g., Benefiber sold by Novartis AG). Other gums such as gum arabic and pectin also have different structures. Other gums include xanthan gum, gellan gum, tara gum, psyllium seed husk gum (psyllium seed husk gum), and locust bean gum.

Waxes are esters of ethylene glycol and two fatty acids, usually present as a hydrophobic liquid that is insoluble in water.

Inulin includes naturally occurring oligosaccharides that belong to a class of carbohydrates known as fructans. They are usually composed of fructose units linked by β - (2-1) glycosidic linkages with a terminal glucose unit. Oligosaccharides are sugar polymers typically containing from three to six component sugars. They are typically found O-linked or N-linked to compatible amino acid side chains or lipid molecules in proteins. Fructooligosaccharides are oligosaccharides composed of short chain fructose molecules.

Food sources of dietary fiber include, but are not limited to, grains, legumes, fruits, and vegetables. Cereals that provide dietary fiber include, but are not limited to, oats, rye, barley, wheat. Legumes that provide fiber include, but are not limited to, peas and beans such as soybeans. Fruits and vegetables that provide a source of fiber include, but are not limited to, apple, orange, pear, banana, berry, tomato, green bean, broccoli, cauliflower, carrot, potato, celery. Vegetable foods such as bran, nuts and seeds (such as flaxseed) are also sources of dietary fiber. Plant parts that provide dietary fiber include, but are not limited to, stems, roots, leaves, seeds, pulp, and bark.

Although dietary fiber is generally derived from plant sources, indigestible animal products such as chitin are also classified as dietary fiber. Chitin is a polysaccharide composed of acetylglucosamine units linked by β (1-4) bonds similar to cellulose bonds.

Dietary fiber sources are generally classified into soluble fiber classes and insoluble fiber classes based on their solubility in water. Both soluble and insoluble fibers are found in plant foods to varying degrees depending on plant characteristics. Although insoluble in water, insoluble fibers have passive hydrophilic characteristics that help increase volume, soften stool, and shorten the transit time of stool solids through the intestinal tract.

Unlike insoluble fibers, soluble fibers are readily soluble in water. Soluble fiber undergoes active metabolic processes by fermentation within the colon, thereby increasing colonic flora and thus increasing the quality of fecal solids. Fermentation of the fiber by colonic bacteria also produces an end product with significant health benefits. For example, fermentation of food quality produces gas and short chain fatty acids. Acids produced during fermentation include butyric, acetic, propionic, and valeric acids with different advantageous properties such as stabilizing blood glucose levels by acting on pancreatic insulin release and providing liver control by glycogen degradation. In addition, fiber fermentation can reduce atherosclerosis by lowering cholesterol synthesis in the liver and reducing blood LDL and triglyceride levels. Acid produced during fermentation lowers colonic pH, thereby protecting the colonic mucosa from the formation of cancer polyps. The reduced colonic pH also increases mineral absorption, improves barrier properties of the colonic mucosal layer, and inhibits inflammation and adhesion irritation. Fermentation of the fiber may also benefit the immune system by stimulating the production of T helper cells, antibodies, leukocytes, splenocytes, cytokinins, and lymphocytes.

Fatty acids

In certain embodiments, the functional ingredient is at least one fatty acid. As used herein, the at least one fatty acid can be a single fatty acid or a plurality of fatty acids as a functional ingredient of the compositions provided herein. Generally, according to particular embodiments of the present invention, the at least one fatty acid is present in the composition in an amount sufficient to promote health and wellness.

As used herein, "fatty acid" refers to any straight chain monocarboxylic acid and includes saturated fatty acids, unsaturated fatty acids, long chain fatty acids, medium chain fatty acids, short chain fatty acids, fatty acid precursors (including omega-9 fatty acid precursors), and esterified fatty acids. As used herein, "long chain polyunsaturated fatty acid" refers to any polyunsaturated carboxylic acid or organic acid having one long aliphatic tail. As used herein, "omega-3 fatty acid" refers to any polyunsaturated fatty acid having one first double bond as a third carbon-carbon bond from the terminal methyl end of its carbon chain. In particular embodiments, the omega-3 fatty acid can include a long chain omega-3 fatty acid. As used herein, "omega-6 fatty acid" refers to any polyunsaturated fatty acid having one first double bond as the sixth carbon-carbon bond from the terminal methyl end of its carbon chain.

Suitable omega-3 fatty acids for use in embodiments of the present invention can be derived from, for example, algae, fish, animals, plants, or combinations thereof. Examples of suitable omega-3 fatty acids include, but are not limited to, linolenic acid, alpha-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, stearidonic acid, eicosatetraenoic acid, and combinations thereof. In some embodiments, suitable omega-3 fatty acids may be provided in fish oils (e.g., herring oil, tuna oil, salmon oil, bonito oil, and cod oil), microalgae omega-3 oils, or combinations thereof. In particular examples, suitable Omega-3 fatty acids may be oils derived from commercially available Omega-3 fatty acids, such as microalgal DHA oil (from marcek, Columbia, MD), Omega pure (from Omega Protein, Houston, TX), dronabinol C-38(Marinol C-38) (from Lipid Nutrition, Channahon, IL), Bonito oil and MEG-3(Bonito oil and MEG-3) (from NS darts marine Nutrition, Dartmouth, NS), Evogel (from symden, holzmington, Germany), oil from tuna or oil from Ocean as sea water, omas, herring, RTP, NC).

Suitable omega-6 fatty acids include, but are not limited to, linoleic acid, gamma-linolenic acid, dihomo-gamma-linolenic acid, arachidonic acid, eicosadienoic acid, docosadienoic acid, adrenic acid, docosapentaenoic acid, and combinations thereof.

Suitable esterified fatty acids for use in embodiments of the present invention may include, but are not limited to, monoacylglycerols containing omega-3 and/or omega-6 fatty acids, diacylglycerols containing omega-3 and/or omega-6 fatty acids, or triacylglycerols containing omega-3 and/or omega-6 fatty acids, and combinations thereof.

Vitamin preparation

In certain embodiments, the functional ingredient is at least one vitamin.

As used herein, the at least one vitamin can be a single vitamin or multiple vitamins that are a functional ingredient of the compositions provided herein. Generally, according to particular embodiments of the present invention, the at least one vitamin is present in the composition in an amount sufficient to promote health and wellness.

Vitamins are organic compounds that the human body needs in small amounts to function properly. The body uses vitamins without destroying them, unlike other nutrients such as carbohydrates and proteins. To date, thirteen vitamins have been recognized, and one or more may be used in the compositions herein. Suitable vitamins include vitamin a, vitamin D, vitamin E, vitamin K, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B12, and vitamin C. Many vitamins also have alternative chemical names, non-limiting examples of which are provided below.

A variety of other compounds have been classified by some authorities as vitamins. These compounds may be referred to as pseudo-vitamins and include, but are not limited to, compounds such as ubiquinone (coenzyme Q10), pangamine, dimethylglycine, taestrile, amygdalin, flavonoids, p-aminobenzoic acid, adenine, adenylic acid, and s-methyl methionine. As used herein, the term vitamin includes pseudovitamins.

In some embodiments, the vitamin is a fat soluble vitamin selected from the group consisting of vitamin a, vitamin D, vitamin E, vitamin K, and combinations thereof.

In other embodiments, the vitamin is a water-soluble vitamin selected from the group consisting of: vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B12, folic acid, biotin, pantothenic acid, vitamin C, and combinations thereof.

Glucosamine

In certain embodiments, the functional ingredient is glucosamine.

Generally, according to particular embodiments of the present invention, glucosamine is present in these compositions in an amount sufficient to promote health and wellness.

Glucosamine (also known as chitosamine) is an amino sugar considered as an important precursor in the biochemical synthesis of glycosylated proteins and lipids. D-glucosamine naturally occurs in cartilage as glucosamine-6-phosphate, which is synthesized from fructose-6-phosphate and glutamine. Glucosamine is, however, also available in other forms, non-limiting examples of which include glucosamine hydrochloride, glucosamine sulfate, N-acetyl-glucosamine, or any other salt form or combination thereof. Glucosamine may be obtained by acid hydrolysis of shells of lobsters, crabs, shrimps, or prawns using methods well known to those of ordinary skill in the art. In one particular embodiment, glucosamine can be derived from fungal biomass containing chitin, as described in U.S. patent publication No. 2006/0172392.

These compositions may further comprise chondroitin sulfate.

Mineral substance

In certain embodiments, the functional ingredient is at least one mineral.

As used herein, the at least one mineral can be a single mineral or multiple minerals that are a functional ingredient of the compositions provided herein. Generally, according to particular embodiments of the present invention, the at least one mineral is present in the composition in an amount sufficient to promote health and wellness.

According to the teachings of the present invention, minerals include inorganic chemical elements that are required by an organism. Minerals are composed of a wide range of compositions (e.g., elements, simple salts, and complex silicates) and also vary widely in crystalline structure. They may be naturally present in foods and beverages, may be added as a supplement, or may be consumed or administered separately from the food or beverage.

Minerals can be classified as either bulk minerals (bulk minerals) which are required in relatively large quantities or trace minerals which are required in relatively small quantities. The bulk minerals generally require an amount of greater than or equal to about 100mg per day and the trace minerals are those minerals that require an amount of less than about 100mg per day.

In particular embodiments of the invention, the mineral is selected from a bulk mineral, a trace mineral, or a combination thereof. Non-limiting examples of host minerals include calcium, chloride, magnesium, phosphorus, potassium, sodium, and sulfur. Non-limiting examples of trace minerals include chromium, cobalt, copper, fluorine, iron, manganese, molybdenum, selenium, zinc, and iodine. Although iodine is generally classified as a trace mineral, it requires greater amounts than other trace minerals and is often classified as a bulk mineral.

In other specific embodiments of the invention, the mineral is a trace mineral that is considered essential for human nutrition, non-limiting examples of which include bismuth, boron, lithium, nickel, rubidium, silicon, strontium, tellurium, tin, titanium, tungsten, and vanadium.

The minerals present herein may be in any form known to one of ordinary skill in the art. For example, in one particular embodiment, the minerals may be in the form of ions having a positive or negative charge. In another embodiment, these minerals may be in their molecular form. For example, sulfur and phosphorus typically occur naturally as sulfates, sulfides, and phosphates.

Preservative

In certain embodiments, the functional ingredient is at least one preservative.

As used herein, the at least one preservative may be a single preservative or a plurality of preservatives as a functional ingredient of the compositions provided herein. Generally, according to particular embodiments of the present invention, the at least one preservative is present in the composition in an amount sufficient to promote health and wellness.

In embodiments of the present invention, the preservative is selected from an antimicrobial agent, an antioxidant, an anti-enzymatic agent, or a combination thereof. Non-limiting examples of antimicrobial agents include sulfites, propionates, benzoates, sorbates, nitrates, nitrites, bacteriocins, salts, sugars, acetic acid, dimethyl dicarbonate (DMDC), ethanol, and ozone.

According to a particular embodiment, the preservative is a sulfite. Sulfites include, but are not limited to, sulfur dioxide, sodium bisulfite, and potassium bisulfite.

According to another embodiment, the preservative is a propionate. Propionates include, but are not limited to, propionic acid, calcium propionate, and sodium propionate.

According to yet another embodiment, the preservative is a benzoate salt. Benzoates include, but are not limited to, sodium benzoate and benzoic acid.

In another embodiment, the preservative is a sorbate salt. Sorbate salts include, but are not limited to, potassium sorbate, sodium sorbate, calcium sorbate, and sorbic acid.

In yet another embodiment, the preservative is a nitrate and/or a nitrite. Nitrates and nitrites include, but are not limited to, sodium nitrate and sodium nitrite.

In yet another embodiment, the at least one preservative is a bacteriocin, such as, for example, nisin.

In another embodiment, the preservative is ethanol.

In yet another embodiment, the preservative is ozone.

Non-limiting examples of anti-enzymatic agents suitable for use as preservatives in particular embodiments of the present invention include ascorbic acid, citric acid, and metal chelating agents such as ethylenediaminetetraacetic acid (EDTA).

Hydrating agent

In certain embodiments, the functional ingredient is at least one hydrating agent.

As used herein, the at least one hydrating agent can be a single hydrating agent or multiple hydrating agents as a functional ingredient of the compositions provided herein. Generally, according to particular embodiments of the present invention, the at least one hydrating agent is present in the composition in an amount sufficient to promote health and wellness.

The hydration products help the body to replace body fluids lost through excretion. For example, body fluids are lost as sweat to regulate body temperature, urine to excrete waste, and water vapor to exchange gases within the lungs. Fluid loss can also occur due to a wide range of external causes, non-limiting examples of which include physical activity, exposure to dry air, diarrhea, vomiting, high fever, shock, blood loss, and hypotension. Diseases that cause fluid loss include diabetes, cholera, gastroenteritis, shigellosis, and yellow fever. Forms of malnutrition that cause fluid loss include excessive consumption of alcohol, electrolyte imbalance, fasting, and rapid weight loss.

In a particular embodiment, the hydration product is a composition that helps the body replace body fluids lost during exercise. Thus, in one particular embodiment, the hydration product is an electrolyte, non-limiting examples of which include sodium, potassium, calcium, magnesium, chloride, phosphate, bicarbonate, and combinations thereof. Suitable electrolytes for use in particular embodiments of the present invention are also described in U.S. Pat. No. 5,681,569, the disclosure of which is expressly incorporated herein by reference. In particular embodiments, the electrolytes are obtained from their respective water-soluble salts. Non-limiting examples of salts for use in particular embodiments include chloride, carbonate, sulfate, acetate, bicarbonate, citrate, phosphate, hydrogen phosphate, tartrate, sorbate, citrate, benzoate, or combinations thereof. In other embodiments, the electrolytes are provided by fruit juice, fruit extract, vegetable extract, tea or tea extract.

In particular embodiments of the invention, the hydration product is a carbohydrate that supplements the energy storage burned by the muscle. Suitable carbohydrates for use in particular embodiments of the present invention are described in U.S. Pat. Nos. 4,312,856, 4,853,237, 5,681,569, and 6,989,171, the disclosures of which are expressly incorporated herein by reference. Non-limiting examples of suitable carbohydrates include monosaccharides, disaccharides, oligosaccharides, complex polysaccharides, or combinations thereof. Non-limiting examples of suitable types of monosaccharides for use in particular embodiments include trioses, tetroses, pentoses, hexoses, heptoses, octoses, and nonoses. Non-limiting examples of specific types of suitable monosaccharides include glyceraldehyde, dihydroxyacetone, erythrose, threose, erythrulose, arabinose, lyxose, ribose, xylose, ribulose, xylulose, allose, altrose, galactose, glucose, gulose, idose, mannose, talose, fructose, psicose, sorbose, tagatose, mannoheptulose, sedoheptulose (sedoheltulose), octulose (octolose), and salivary sugar (sialose). Non-limiting examples of suitable disaccharides include sucrose, lactose, and maltose. Non-limiting examples of suitable oligosaccharides include sucrose, maltotriose, and maltodextrin. In other embodiments, the carbohydrate is provided by corn syrup, beet sugar, cane sugar, fruit juice, or tea.

In another embodiment, the hydration is a flavanol that provides cellular rehydration. Flavanols are a class of natural substances present in plants and typically comprise a 2-phenylbenzopyranone molecular backbone attached to one or more chemical moieties. Non-limiting examples of suitable flavanols for use in specific embodiments of the present invention include catechin, epicatechin, gallocatechin, epigallocatechin, epicatechin gallate, epigallocatechin 3-gallate, theaflavin 3-gallate, theaflavin 3 '-gallate, theaflavin 3, 3' -gallate, thearubigin, or combinations thereof. Several common sources of flavanols include tea, fruits, vegetables, and flowers. In preferred embodiments, the flavanols are extracted from green tea.

In a particular embodiment, the hydration product is a glycerol solution that enhances exercise endurance. The uptake of a solution containing glycerol has been shown to provide a number of beneficial physiological effects, such as enlarged blood volume, reduced heart rate, and reduced rectal temperature.

Probiotics/prebiotics

In certain embodiments, the functional ingredient is selected from at least one probiotic, prebiotic, and combinations thereof.

As used herein, the at least one probiotic or prebiotic may be a single probiotic or prebiotic or a plurality of probiotics or prebiotics as a functional ingredient of the compositions provided herein. Generally, according to particular embodiments of the present invention, the at least one probiotic, prebiotic, or combination thereof is present in the composition in an amount sufficient to promote health and wellness.

In accordance with the teachings of the present invention, probiotics include microorganisms that are beneficial to health when consumed in effective amounts. Ideally, probiotics favorably impact the human body's naturally occurring gastrointestinal microflora and confer health benefits in addition to nutrition. Probiotics may include, without limitation, bacteria, yeasts, and fungi.

In accordance with the teachings of the present invention, prebiotics are compositions that promote the growth of beneficial bacteria in the intestine. The prebiotic substance may be consumed by one of the associated probiotics or otherwise help to keep the associated probiotic alive or stimulate its growth. When consumed in effective amounts, probiotics also beneficially affect the human body's naturally occurring gastrointestinal microbiota disorders thus conferring health benefits in addition to mere nutrition. Prebiotic foods enter the colon and serve as substrates for endophytes, indirectly providing energy, metabolic substrates, and essential micronutrients to the host. Digestion and absorption of the body's prebiotic food is dependent on bacterial metabolic activity, which rescues the host's energy from nutrients that escape digestion and absorption in the small intestine.

According to a particular embodiment, the probiotic is a beneficial microorganism that favorably affects the human body's naturally occurring gastrointestinal microflora and imparts health benefits in addition to nutrition. Examples of probiotics include, but are not limited to, bacteria of the genus lactobacillus (lactobacillus), bifidobacterium (bifidobacterium), streptococcus (streptococcus), or combinations thereof that impart beneficial effects to humans.

In a particular embodiment of the invention, the at least one probiotic is selected from the genus lactobacillus. Lactobacillus (i.e., lactobacillus bacteria, followed by "l.") has been used as a food method and to promote human health for centuries. Non-limiting examples of lactobacillus species visible in the human gastrointestinal tract include lactobacillus acidophilus (l.acidophilus), lactobacillus casei (l.casei), lactobacillus fermentum (l.fermentum), lactobacillus salivarius (l.saliva roes), lactobacillus brevis (l.brevis), lactobacillus reicheri (l.leichmanii), lactobacillus plantarum (l.plantatarum), lactobacillus cellobiosus (l.cellobiosus), lactobacillus reuteri (l.reuteri), lactobacillus rhamnosus (l.rhamnosus), lactobacillus GG (l.gg), lactobacillus bulgaricus (l.bulgaricus), and lactobacillus thermophilus (l.thermophilus).

According to other particular embodiments of the invention, the probiotic is selected from the genus bifidobacterium. Bifidobacteria are also known to exert a beneficial effect on human health through carbohydrate metabolism to produce short chain fatty acids (e.g., acetic, propionic, and butyric acids), lactic, and formic acids. Non-limiting species of bifidobacteria found in the human gastrointestinal tract include bifidobacterium infantis (b.angulus), bifidobacterium animalis (b.animalis), bifidobacterium asteroidea (b.asteroides), bifidobacterium bifidum (b.bifidum), bifidobacterium breve (b.boum), bifidobacterium catenulatum (b.catenulatum), bifidobacterium minium (b.catenulatum), bifidobacterium minor swine (b.chloroerunum), bifidobacterium corynebacterium (b.coeyneforme), bifidobacterium striiformis (b.cuniculinum), bifidobacterium odonum (b.dentium), bifidobacterium gaurea (b.gallinarum), bifidobacterium gallinarum (b.gallinarum), bifidobacterium chrysanthum (b.indicum), bifidobacterium longum (b.longum), bifidobacterium magenum (b.magnum), bifidobacterium ruminans (b.ruminodes), bifidobacterium merum (b.merum), bifidobacterium minium (b.carinatum), bifidobacterium longum (b.carinatum) B. scardovii, bifidobacterium ape (b.simiae), bifidobacterium microsecond (b.subtile), bifidobacterium thermophilum (b.thermophilum), bifidobacterium urosum (b.urinalis), and certain species of bifidobacterium.

According to other particular embodiments of the invention, the probiotic is selected from the genus streptococcus. Streptococcus thermophilus is a gram-positive facultative anaerobe. It is classified as a lactic acid bacterium and is commonly found in milk and dairy products, and is used to produce yogurt. Other non-limiting probiotic species of this bacterium include Streptococcus salivarius (Streptococcus salivarius) and Streptococcus cremoris (Streptococcus cremoris).

The probiotics which can be used according to the invention are well known to the person skilled in the art. Non-limiting examples of food products comprising probiotics include yogurt, german kimchi, goat cheese, korean kimchi, fermented vegetables, and other food products containing microbial elements that favorably affect the host animal by improving intestinal micro-balance.

According to embodiments of the present invention, prebiotics include, without limitation, mucopolysaccharides, oligosaccharides, polysaccharides, amino acids, vitamins, nutrient precursors, proteins, and combinations thereof.

According to a particular embodiment of the invention, the prebiotic is selected from dietary fibers, including, without limitation, polysaccharides and oligosaccharides. These compounds have the ability to increase the number of probiotics which results in the benefits given by these probiotics. Non-limiting examples of oligosaccharides classified as prebiotics according to particular embodiments of the present invention include fructooligosaccharides, inulin, isomaltooligosaccharides, lactitol (lactilol), lactulose oligosaccharides, lactulose, pyrodextrins, soy oligosaccharides, transgalactooligosaccharides, and xylooligosaccharides.

According to other particular embodiments of the invention, the prebiotic is an amino acid. While many known prebiotics break down to provide carbohydrates for probiotics, some probiotics also require amino acids to provide nutrients.

Prebiotics are found naturally in a variety of foods, including, without limitation, bananas, berries, asparagus, garlic, wheat, oats, barley (and other whole grains), linseed, tomato, jerusalem artichoke, onion and chicory, vegetable leaves (green) (e.g., dandelion tender leaf, spinach, kale leaf, sugar beet, kale, mustard leaf, turnip leaf), and beans (e.g., lentils, kidney beans, chickpeas, navy beans, white beans, black beans).

Weight management agent

In certain embodiments, the functional ingredient is at least one weight management agent.

As used herein, the at least one weight management agent can be a single weight management agent or multiple weight management agents as a functional ingredient of the compositions provided herein. Generally, according to particular embodiments of the present invention, the at least one weight management agent is present in the composition in an amount sufficient to promote health and wellness.

As used herein, "weight management agent" includes an appetite suppressant and/or thermogenic agent. As used herein, the phrases "appetite suppressant", "appetite-satiating composition", "satiety agent", and "satiety composition" are synonymous. The phrase "appetite suppressant" describes macronutrients, herbal extracts, exogenous hormones, anorectics, drugs and combinations thereof that suppress, reduce or otherwise shorten a person's appetite when delivered in an effective amount. The phrase "thermogenic agent" describes macronutrients, herbal extracts, exogenous hormones, anorectic agents, drugs and combinations thereof that stimulate or otherwise enhance thermogenesis or metabolism in humans when delivered in effective amounts.

Suitable weight management agents include macronutrients selected from the group consisting of: protein, carbohydrate, dietary fat, and combinations thereof. Consumption of protein, carbohydrates, and dietary fat stimulates the release of peptides with appetite suppression. For example, consumption of protein and dietary fat stimulates the release of the gastrointestinal hormone cholecystokinin (CCK), while consumption of carbohydrate and dietary fat stimulates the release of glucagon-like peptide 1 (GLP-1).

Suitable macronutrient management agents also include carbohydrates. Carbohydrates generally include sugars, starches, cellulose and gums that the body converts to glucose for energy. Carbohydrates are generally divided into two classes, digestible carbohydrates (e.g., monosaccharides, disaccharides, and starches) and non-digestible carbohydrates (e.g., dietary fibers). Studies have shown that carbohydrates that are not digestible in the small intestine and complex polymer carbohydrates with reduced absorption and digestibility stimulate physiological responses that inhibit food intake. Thus, the carbohydrates presented herein desirably include indigestible carbohydrates or carbohydrates with reduced digestibility. Non-limiting examples of such carbohydrates include polydextrose; inulin; monosaccharide derived polyols such as erythritol, mannitol, xylitol, and sorbitol; disaccharide derived alcohols such as isomalt, lactitol, and maltitol; and hydrogenated starch hydrolysates. Carbohydrates are described in more detail herein below.

In another embodiment, the weight management agent is a dietary fat. Dietary fat is a lipid comprising a combination of saturated and unsaturated fatty acids. Polyunsaturated fatty acids have been shown to have greater satiety capacity than monounsaturated fatty acids. Thus, the dietary fats presented herein desirably include polyunsaturated fatty acids, non-limiting examples of which include triacylglycerols.

In one embodiment, the weight management agent is a herbal extract. Extracts from various types of plants have been identified as having appetite suppressant properties. Non-limiting examples of plants whose extracts have appetite-suppressing properties include plants of the genera Huolunia (Hoodia), Trichocaulon (Trichocaulon), Caralluma, Leoparda (Stapelia), Obelina (Orbea), Asclepias (Asclepias), and Camellia (Camellia). Other examples include extracts derived from Gymnema Sylvestre, Kola Nut, lime (Citrus aurantium), Yerba Mate (Yerba Mate), gardnia griffiana (Griffonia silcifolia), Guarana (Guarana), myrrha (myrrh), carageenan (gum Lipid), and blackcurrant seed oil (black current seed oil).

The herbal extract may be prepared from any type of plant material or plant biomass. Non-limiting examples of plant material and biomass include stems, roots, leaves, dried powders obtained from plant material, and sap or dried sap. Herbal extracts are typically prepared by extracting sap from the plant and then spray drying the sap. Alternatively, a solvent extraction procedure may be used. After the initial extraction, it may be desirable to further fractionate the initial extract (e.g., by column chromatography) in order to obtain an herbal extract with enhanced activity. Such techniques are well known to those of ordinary skill in the art.

In a particular embodiment, the herbal extract is a plant derived from the subfamily of the genus Hoodia, the species of which include H.alstonii, H.currorii, H.dregii, Hoodia gordonii (H.flava), Hoodia cactus (H.gordonii), H.jutatae, H.mossapiensis, Hoodia sanguisorba (H.oficinalis), H.parviflora, Hoodia clarke (H.pedicellata), H.pilifera, H.ruschii, and H.triebneri. The plants of the genus Hoodia are carnivorous plants native to south Africa. A Flexidia sterol glycoside, designated P57, is believed to be responsible for the appetite suppressant effect of Flexidia species.

In another embodiment, the herbal extract is derived from a plant of the genus calophyllum (genus Caralluma), the species of which include cactus indica (c.indica), c.fimbriata, c.attenuate, cactus broussii (c.tubericula), opuntia ficus-indica (c.edulis), opuntia microphylla (c.adscenses), c.stalagmifera, opuntia umbellata (c.umbellate), c.penicilata, c.sessile, c.russeliana, c.retrospec, c.arabilica, and c.lasiantha. The caralluma plant belongs to the same Asclepiadaceae of the subfamily as the genus Hoodia. Caralluma palmata is a short, erect and fleshy plant native to india with medical properties such as appetite suppression, which are generally attributed to glycosides belonging to the glycosidoprogestational group, non-limiting examples of which include tumoural glycoside (caratuberside) a, tumoural glycoside B, bunoloside (boucheroside) I, bunoloside II, bunoloside III, bunoloside IV, bunoloside V, bunoloside VI, bunoloside VII, bunoloside VIII, bunoloside IX, and bunoloside X.

In another embodiment, the at least one herbal extract is derived from a plant of the genus arhat. Plants of the genus arhat are succulent plants usually native to south africa, similar to the genus geotrichum, and include morehringer (t.piliferum) and t.offisile.

In another embodiment, the herbal extract is derived from a plant of the genus leopard or the genus obesia, and their species include sea anemone long beard (s.gigantean) and variegated leopard flower (o.variegate), respectively. Both the leopard and obedienia plants belong to the same sub-family Asclepiadaceae as the fire geosub-genus. Without wishing to be bound by any theory, it is believed that these compounds exhibiting appetite suppressing activity are saponins, such as pregnane glycosides, which include variegated leopard kadsin (stavaroside) A, B, C, D, E, F, G, H, I, J, and K.

In another embodiment, the herbal extract is derived from a plant of the genus milkweed. The plant of genus Asclepiadaceae also belongs to the Asclepiadaceae family. Non-limiting examples of plants of the genus milkweed include milkweed (a. incarnate), yellowhorn milkweed (a. curassayica), syrian milkweed (a. syriaca), and willow milkweed (a. tuberose). Without wishing to be bound by any theory, it is believed that these extracts contain steroids such as pregnane glycosides and pregnane aglycones which have an appetite suppressant effect.

In one embodiment, the weight management agent is an exogenous hormone having weight management properties. Non-limiting examples of such hormones include CCK, peptide YY, ghrelin, bombesin and Gastrin Releasing Peptide (GRP), enterostatin, apolipoprotein A-IV, GLP-1, amylin, somatostatin, and leptin.

In another embodiment, the weight management agent is a drug. Non-limiting examples include phentermine, diethylpropion, phendimetrazine, sibutramine, rimonabant, oxyntomodulin, fluoxetine hydrochloride, ephedrine, phenylethylamine, or other irritants.

Osteoporosis management agent

In certain embodiments, the functional ingredient is at least one osteoporosis management agent.

As used herein, the at least one osteoporosis management agent may be a single osteoporosis management agent or multiple osteoporosis management agents as a functional ingredient of the compositions provided herein. Generally, according to particular embodiments of the present invention, the at least one osteoporosis management agent is present in the composition in an amount sufficient to promote health and wellness.

Osteoporosis is a skeletal disorder of compromised bone strength that causes an increased risk of bone fracture. Osteoporosis is generally characterized by a reduction in Bone Mineral Density (BMD), destruction of bone micro-structures, and changes in the amount and type of non-collagenous proteins in bone.

In certain embodiments, the osteoporosis management agent is at least one calcium source. According to a particular embodiment, the calcium source is any compound containing calcium, including salt complexes, dissolved substances, and other forms of calcium. Non-limiting examples of calcium sources include amino acid chelated calcium, calcium carbonate, calcium oxide, calcium hydroxide, calcium sulfate, calcium chloride, calcium phosphate, calcium hydrogen phosphate, calcium dihydrogen phosphate, calcium citrate, calcium malate, calcium citrate malate, calcium gluconate, calcium tartrate, calcium lactate, dissolved substances thereof, and combinations thereof.

According to a particular embodiment, the osteoporosis management agent is a source of magnesium. The magnesium source is any compound containing magnesium, including salt complexes, dissolved species, and other forms of magnesium. Non-limiting examples of magnesium sources include magnesium chloride, magnesium citrate, magnesium glucoheptonate, magnesium gluconate, magnesium lactate, magnesium hydroxide, magnesium picolinate (magnesium picoliate), magnesium sulfate, dissolved species thereof, and mixtures thereof. In another embodiment, the magnesium source comprises an amino acid chelated magnesium or creatine chelated magnesium.

In other embodiments, the osteoporosis agent is selected from vitamin D, C, K, precursors thereof, and/or beta-carotene, and combinations thereof.

Various plants and plant extracts have also been identified as effective for the prevention and treatment of osteoporosis. Without wishing to be bound by any theory, it is believed that these plants and plant extracts stimulate osteogenic proteins and/or inhibit bone resorption, thereby promoting bone regeneration and strength. Non-limiting examples of suitable plants and plant extracts as osteoporosis management agents include the species Taraxacum (Taraxacum) and Amelanchier (Amelanchier) as disclosed in U.S. patent publication No. 2005/0106215, and the species Lindera (Lindera), Artemisia (Artemisia), Acorus (Acorus), Carthamus (Carthamus), Carum (Carum), Cnidium (Cnidium), Curcuma (Curcuma), Cyperus (Cyperus), Juniperus (Juniperus), Prunus (Prunus), Iris (Iris), Cichorium (Cichorium), Potentilla (Dodonaea), Epimedium (Epimedium), Vibrio (Erigonoum), Glycine (Soya), Mentha (Mexico), Ocimum (Ocimum), Thymus (Ocimum), Plantago (Ocimum), Hordeum (Salvia), Hordeum (Thulium), and Hibiscus) as disclosed in U.Patrinia (2005/0079232, Species of the genera rosmarinus (Rosemarinus), Rhus (Rhus), and dill (Anethum).

Phytoestrogen

In certain embodiments, the functional ingredient is at least one phytoestrogen.

As used herein, the at least one phytoestrogen may be a single phytoestrogen or multiple phytoestrogens as a functional ingredient of the compositions provided herein. Generally, according to particular embodiments of the present invention, the at least one phytoestrogen is present in the composition in an amount sufficient to promote health and wellness.

Phytoestrogens are compounds found in plants that can typically be delivered to the human body by ingestion of plants or plant parts bearing these phytoestrogens. As used herein, "phytoestrogen" refers to any substance that causes an estrogen-like effect to any degree when introduced into the body. For example, a phytoestrogen can bind to estrogen receptors in the body and has little estrogen-like effect.

Examples of suitable phytoestrogens for use in embodiments of the present invention include, but are not limited to, isoflavones, stilbenes, lignans, resorcylic acid lactone (resorcylic acid lactone), coumarin, coumestrol (coumestan), coumestrol (coumestrol), equol, and combinations thereof. Suitable sources of phytoestrogens include, but are not limited to, whole grains, cereals, fibers, fruits, vegetables, black cohosh, agave roots, black currants, cherry leaf pods, cherry berries, spastic bark, angelica roots, devil's claw roots, false stringy roots (false unicorn root), ginseng roots, sorghead, licorice juice, radicle grasses, motherwort, peony roots, raspberry leaves, rosaceous plants, sage leaves, sargentglomus seeds, wild yam roots, flowering yarrow, legumes, soybeans, soybean products (e.g., miso, soybean flour, soy milk, soybean nuts, soybean protein isolates, marjoram (tempen), or tofu), chickpeas, nuts, lentils, seeds, clovers, red clovers, dandelion leaves, dandelion roots, lupulus seeds, green tea, hops, red beans, flaxseed, linseed, red cohosh, black cohosh, agave roots, black currant roots, black currants, cherry root, ginseng, sage root, ginseng, garlic, onion, linseed, borage, tuberous root milkweed (Butterfly weed), caraway, glossy privet tree, vitex, jujube, dill, fennel seed, centella asiatica, silybum marianum, mentha labiata, pomegranate, artemisia annua, bean flour, chrysanthemum indicum, kudzu root (kudzu root), and the like, and combinations thereof.

Isoflavones belong to the group of plant nutrients known as polyphenols. Generally, polyphenols (also known as "polyphenols") are a group of chemical substances found in plants, characterized by the presence of more than one phenol group per molecule.

Suitable phytoestrogen isoflavones according to embodiments of the present invention include genistein, daidzein, glycitein, biochanin A, formononetin, their respective naturally occurring glycosides and glycoside conjugates, matairesinol, secoisolariciresinol, intestinal diesters, intestinal glycols, plant tissue proteins, and combinations thereof.

Suitable sources of isoflavones for use in embodiments of the present invention include, but are not limited to, soybeans, soybean products, legumes, alfalfa sprouts (alfalfalfa spits), chickpeas, peanuts, and red clover.

Long chain primary aliphatic saturated alcohols

In certain embodiments, the functional ingredient is at least one long chain primary aliphatic saturated alcohol.

As used herein, the at least one long chain aliphatic primary saturated alcohol may be a single long chain aliphatic primary saturated alcohol or a plurality of long chain aliphatic primary saturated alcohols as a functional ingredient of the compositions provided herein. Generally, in accordance with a particular embodiment of the present invention, the at least one long chain aliphatic primary saturated alcohol is present in the composition in an amount sufficient to promote health and wellness.

Long chain aliphatic saturated primary alcohols are a diverse group of organic compounds. The term "alcohol" refers to the fact that: these compounds are characterized by a hydroxyl group (-OH) bonded to a carbon atom. The term primary refers to the fact that: in these compounds, the carbon atom to which the hydroxyl group is bonded to only one additional carbon atom. The term saturation refers to the fact that: these compounds are characterized by the absence of carbon-carbon pi bonds. The term aliphatic refers to the fact that: the carbon atoms in these compounds are linked together in a straight or branched chain rather than in a ring. The term long chain refers to the fact that: the number of carbon atoms in these compounds is at least 8 carbons.

Non-limiting examples of specific long chain aliphatic saturated primary alcohols useful in specific embodiments of the present invention include 8 carbon 1-octanol, 9 carbon 1-nonanol, 10 carbon 1-decanol, 12 carbon 1-dodecanol, 14 carbon 1-tetradecanol, 16 carbon 1-hexadecanol, 18 carbon 1-octadecanol, 20 carbon 1-eicosanol, 22 carbon 1-docosanol, 24 carbon 1-tetracosanol, 26 carbon 1-hexacosanol, 27 carbon 1-heptacosanol, 28 carbon 1-octacosanol (octacosanol), 29 carbon 1-nonacosanol, 30 carbon 1-triacontanol, 32 carbon 1-triacontanol, and 34 carbon 1-triacontanol.

In a particularly desirable embodiment of the present invention, the long chain primary aliphatic saturated alcohol is polycosanol. Polycosanol is a term referring to a mixture of long chain aliphatic primary saturated alcohols consisting essentially of: 28C 1-octacosanol and 30C 1-triacontanol, and lower concentrations of other alcohols such as 22C 1-docosanol, 24C 1-tetracosanol, 26C 1-hexacosanol, 27C 1-heptacosanol, 29C 1-nonacosanol, 32C 1-tridecanol, and 34C 1-tridecanol.

The long chain primary aliphatic saturated alcohols are derived from natural fats and oils. They may be obtained from these sources by using extraction techniques well known to those of ordinary skill in the art. Polycosanols can be isolated from a variety of plants and materials, including sugarcane (sugarcane of sugarcane, sugar cane), yam (e.g., Dioscorea opposita), rice bran (e.g., Oryza sativa), and beeswax. Polycosanol may be obtained from such sources by using extraction techniques well known to those of ordinary skill in the art. A description of such extraction techniques can be found in U.S. patent application No. 2005/0220868, the disclosure of which is expressly incorporated by reference.

Plant sterol

In certain embodiments, the functional ingredient is at least one phytosterol, phytostanol, or combination thereof.

Generally, according to particular embodiments of the present invention, the at least one phytosterol, phytostanol, or combination thereof is present in the composition in an amount sufficient to promote health and wellness.

As used herein, the phrases "stanol", "stanol of a plant" and "phytostanol" are synonymous.

Phytosterols and stanols are found naturally in small amounts in many fruits, vegetables, nuts, seeds, grains, legumes, vegetable oils, bark, and other plant sources. Although people normally consume phytosterols and stanols daily, the amounts consumed are not sufficient to have significant cholesterol lowering effects or other health benefits. Therefore, it is desirable to supplement foods and beverages with phytosterols and stanols.

Sterols are a subgroup of steroids having a hydroxyl group at C-3. Generally, phytosterols have one double bond within the sterol core, such as cholesterol; however, the phytosterols may also comprise a substituted side chain (R) at C-24, such as ethyl or methyl, or an additional double bond. The structure of phytosterols is well known to those skilled in the art.

At least 44 naturally occurring phytosterols have been found and they are typically derived from plants such as corn, soybean, wheat, and tung oil; however, they may also be produced synthetically to form compositions identical to those of nature or having properties similar to those of naturally occurring phytosterols. Non-limiting examples of phytosterols that are well known to those of ordinary skill in the art, in accordance with particular embodiments of the present invention, include 4-desmethyl sterols (e.g., beta-sitosterol, campesterol, stigmasterol, brassicasterol, 22-dehydrobrassicasterol, and Δ 5-avenasterol), 4-monomethyl sterols, and 4, 4-dimethyl sterols (triterpene alcohols) (e.g., cycloartenol, 24-methylenecycloartanol, and cyclostanol (cyclobranol)).

As used herein, the phrases "stanol," "phytostanol," and "phytostanol" are synonymous. Phytostanols are saturated sterols that are present in nature only in minute amounts and can also be produced synthetically, for example by hydrogenation of phytosterols. Non-limiting examples of phytostanols according to particular embodiments of the present invention include beta-sitostanol, campestanol, cycloartenol, and saturated forms of other triterpene alcohols.

Phytosterols and phytostanols, as used herein, include a variety of isomers such as the alpha and beta isomers (e.g., alpha-sitosterol and beta-sitostanol, which include one of the most effective phytosterols and phytostanols, respectively, for lowering serum cholesterol in mammals).

The phytosterols and phytostanols of the present invention may also be in their ester form. Suitable methods for obtaining esters of phytosterols and phytostanols are well known to those of ordinary skill in the art and are disclosed in U.S. patent nos. 6,589,588, 6,635,774, 6,800,317, and U.S. patent publication No. 2003/0045473, the disclosures of which are incorporated herein by reference in their entirety. Non-limiting examples of suitable esters of phytosterols and phytostanols include sitosterol acetate, sitosterol oleate, stigmasterol oleate, and their corresponding phytostanol esters. The phytosterols and phytostanols of the present invention may also include derivatives thereof.

Generally, the amount of functional ingredient in the composition will vary widely depending on the particular composition and functional ingredient desired. One of ordinary skill in the art will readily determine the appropriate amounts of the functional ingredients for each composition.

In one embodiment, a method for preparing a composition comprises combining rebaudioside I and at least one sweetener and/or additive and/or functional ingredient.

Consumer product

In one embodiment, the composition of the present invention is a consumable comprising rebaudioside I or a consumable containing a composition comprising rebaudioside I (e.g., a sweetener composition).

Rebaudioside I or compositions comprising it may be incorporated in any known edible or oral compositions (referred to herein as "consumables"), such as, for example, pharmaceutical compositions, edible gel mixes and compositions, dental compositions, food products (confections, condiments, chewing gums, cereal compositions, baked goods, dairy products, and tabletop sweetener compositions), beverages, and beverage products.

As used herein, consumable product refers to a substance that comes into contact with the mouth of a human or animal, including substances that are ingested and subsequently expelled from the mouth and substances that are drunk, eaten, swallowed or otherwise ingested, and are healthy for human or animal consumption when used in a generally acceptable range.

For example, the beverage is a consumable product. The beverage can be sweetened or unsweetened prior to addition of rebaudioside I or a composition comprising rebaudioside I. Rebaudioside I, or compositions comprising rebaudioside I, can be added to a beverage or beverage base to sweeten the beverage or enhance its existing sweetness or flavor.

In one embodiment, the invention is a consumable comprising rebaudioside I. The concentration of rebaudioside I in the consumable can be above, at, or below its threshold sweetness concentration.

In a particular embodiment, the invention is a beverage comprising rebaudioside I. The concentration of rebaudioside I in the beverage may be above, at, or below its threshold sweetness concentration.

The consumable product may optionally comprise additives, additional sweeteners, functional ingredients, and combinations thereof, as described herein. Any of the additives, additional sweeteners, and functional ingredients described above may be present in the consumable.

Pharmaceutical composition

In one embodiment, the present invention is a pharmaceutical composition comprising a pharmaceutically active substance and rebaudioside I.

In another embodiment, the present invention is a pharmaceutical composition comprising a pharmaceutically active substance and a composition comprising rebaudioside I.

Rebaudioside I or compositions comprising rebaudioside I can be present in the pharmaceutical composition as an excipient material that can mask the bitter or other undesirable taste of a pharmaceutically active substance or another excipient material. The pharmaceutical composition may be in the form of: tablets, capsules, liquids, aerosols, powders, effervescent tablets or powders, syrups, emulsions, suspensions, solutions, or any other form to provide a pharmaceutical composition to a patient. In particular embodiments, the pharmaceutical composition may be in a form for oral administration, buccal administration, sublingual administration, or any other route of administration known in the art.

As referred to herein, "pharmaceutically active substance" means any drug, pharmaceutical agent, medicament, prophylactic, therapeutic, or other substance that has biological activity. As referred to herein, an "excipient material" refers to any inactive substance that acts as a vehicle for an active ingredient, such as any material that facilitates handling, stability, dispersibility, wettability, and/or release kinetics of a pharmaceutically active substance.

Suitable pharmaceutically active substances include, but are not limited to, agents for the gastrointestinal or digestive system, agents for the cardiovascular system, agents for the central nervous system, agents for pain or consciousness, agents for musculoskeletal disorders, agents for the eye, agents for the ear, nose and oropharynx, agents for the respiratory system, agents for endocrine problems, agents for the reproductive or urinary system, agents for contraception, agents for obstetrics and gynecology, agents for skin, agents for infection and infection, agents for immunology, agents for allergic disorders, agents for nutrition, agents for hematologic neoplastic diseases, agents for diagnosis, agents for euthanasia, or agents for other biological functions or disorders. Examples of suitable pharmaceutically active substances for use in embodiments of the present invention include, but are not limited to, antacids, reflux inhibitors, antiflatulents, antihyptamines, proton pump inhibitors, cytoprotectants, prostaglandin analogs, laxatives, antispasmodics, antidiarrheals, bile acid sequestrants, opioids, beta-blockers, calcium channel blockers, diuretics, cardiac glycosides, antiarrhythmics, nitrates, antianginals, vasoconstrictors, vasodilators, peripheral vascular activators, ACE inhibitors, angiotensin receptor blockers, alpha blockers, anticoagulants, heparin, antiplatelet agents, fibrinolytics, antihyphilics, antihyperlipidemic agents, statins, hypnotics (antihyptics), anesthetics, antipsychotics, antidepressants, antiemetics, anticonvulsants, antiepileptics, anxiolytics, Barbiturates, dyskinetic drugs, stimulants, benzodiazepines, cyclic pyrrolidones, dopamine antagonists, antihistamines, cholinergics, anticholinergics, emetics, cannabinoids, analgesics, muscle relaxants, antibiotics, aminoglycosides, antivirals, antifungals, anti-inflammatories, anti-glaucoma drugs, sympathomimetics, steroids, cerumen dissolvents (cerumenolytics), bronchodilators, NSAIDS, antitussives, mucolytics, decongestants, corticosteroids, androgens, antiandrogens, gonadotropins, growth hormones, insulins, antidiabetics, thyroid hormones, calcitonin, bisphosphonates, antidiuretic hormone analogues, basifying agents, quinolones, anticholinesterases, sildenafil, oral contraceptives, hormone replacement therapies, bone regulators, follicle stimulating hormones, luteinizing hormones, linolenic acid (galenic acid), Progestogens, dopamine agonists, estrogens, prostaglandins, gonadotropin releasing factor, clomiphene, tamoxifen, diethylstilbestrol, anti-leprosy, anti-tubercular, anti-malarial, anthelmintic, antiprotozoal, antisera, vaccines, interferons, tonic drugs, vitamins, cytotoxic drugs, sex hormones, aromatase inhibitors, somatostatin inhibitors, or similar types of substances, or combinations thereof. Such components are generally considered safe (GRAS) and/or are U.S. Food and Drug Administration (FDA) approved.

The pharmaceutically active substance is present in the pharmaceutical composition in a wide range of amounts depending on the particular pharmaceutically active agent used and its intended application. An effective dose of any of the pharmaceutically active substances described herein can be readily determined by using conventional techniques and by observing results obtained under similar circumstances. In determining an effective dose, a variety of factors are considered, including, but not limited to, the type of patient; its size, age and general health; the particular disease involved; the degree of involvement or the severity of the disease; individual patient response; the specific pharmaceutically active agent administered; a mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; and the combined use of the medicines. The pharmaceutically active substance is contained in a pharmaceutically acceptable carrier, diluent or excipient in an amount sufficient for delivery to the patient, which is an in vivo effective amount of the pharmaceutically active substance, without serious toxic effects when used in generally acceptable amounts. Thus, suitable amounts can be readily determined by one skilled in the art.

According to particular embodiments of the present invention, the concentration of the pharmaceutically active substance in the pharmaceutical composition will depend on the rate of absorption, inactivation, and excretion of the drug, as well as other factors known to those skilled in the art. It should be noted that dosage values will vary as the severity of the condition diminishes. It is to be further understood that for any particular subject, the particular dosage regimen will be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the pharmaceutical composition, and that the dosage ranges described herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions. The pharmaceutically active substance may be administered once, or may be divided into a plurality of smaller doses to be administered at different time intervals.

The pharmaceutical composition may also comprise other pharmaceutically acceptable excipient materials. Examples of suitable excipient materials for embodiments of the invention include, but are not limited to, antiadherents, binders (e.g., microcrystalline cellulose, tragacanth or gelatin), coatings, disintegrants, fillers, diluents, emollients, emulsifiers, flavoring agents, colorants, adjuvants, lubricants, functional agents (e.g., nutrients), viscosity modifiers, bulking agents, glidants (e.g., colloidal silicon dioxide), surfactants, osmotic agents, diluents, or any other inactive ingredient, or combinations thereof. For example, the pharmaceutical composition of the invention may comprise an excipient material selected from the group consisting of: calcium carbonate, coloring agents, whitening agents, preservatives and flavors, triacetin, magnesium stearate, hydrogenated vegetable oil (sterote), natural or artificial flavors, essential oils, plant extracts, fruit essences, gelatin, or combinations thereof.

The excipient material of the pharmaceutical composition may optionally include other artificial or natural sweeteners, bulk sweeteners, or combinations thereof. Bulk sweeteners include both caloric and non-caloric compounds. In one embodiment, the additive is used as a bulk sweetener. Non-limiting examples of bulk sweeteners include sucrose, dextrose, maltose, dextrin, dried invert sugar, fructose, high fructose corn syrup, levulose, galactose, corn syrup solids, tagatose, polyols (e.g., sorbitol, mannitol, xylitol, lactitol, erythritol, and maltitol), hydrogenated starch hydrolysates, isomalt, trehalose, and mixtures thereof. In particular embodiments, bulk sweeteners are present in the pharmaceutical composition in a wide range of amounts depending on the desired sweetness. Suitable amounts of the two sweeteners will be readily discernible to those skilled in the art.

Edible gel mixture and edible gel composition

In one embodiment, the invention is an edible gel or edible gel blend comprising rebaudioside I. In another embodiment, the invention is an edible gel or an edible gel blend comprising a composition comprising rebaudioside I.

Edible gels are gels that can be eaten. A gel is a colloidal system in which a network of particles spans the volume of a liquid medium. Although gels are primarily composed of liquids and therefore exhibit densities similar to liquids, gels have a structural coherence of solids due to a network of particles spanning the liquid medium. For this reason, gels generally appear as solid, jelly-like materials. Gels can be used in a variety of applications. For example, gels may be used in foods, paints and adhesives.

Non-limiting examples of edible gel compositions for use in particular embodiments include gel snacks, puddings, jellies, pastes, sponge cakes, aspics (aspics), marshmallows, gummy creamy candies, and the like. Edible gel mixtures are typically powdered or granular solids to which a fluid may be added to form an edible gel composition. Non-limiting examples of fluids for use in particular embodiments include water, dairy fluids, dairy-like fluids, juices, alcohols, alcoholic beverages, and combinations thereof. Non-limiting examples of dairy fluids that may be used in particular embodiments include milk, yogurt, cream, fluid whey, and mixtures thereof. Non-limiting examples of dairy-like fluids that may be used in particular embodiments include, for example, soy milk and non-dairy coffee whiteners. Because edible gel products found on the market are typically sweetened with sucrose, it is desirable to sweeten an edible gel with a replacement sweetener to provide a low or non-caloric replacement.

As used herein, the term "gelling component" refers to any material that can form a colloidal system within a liquid medium. Non-limiting examples of gelling components useful in particular embodiments include gelatin, alginates, carrageenan, gums, pectins, konjac gum, agar, edible acids, rennet, starch derivatives, and combinations thereof. It is well known to those of ordinary skill in the art that the amount of gelling ingredients used in an edible gel mixture or edible gel composition will vary appropriately depending on a variety of factors, such as the particular gelling ingredients used, the particular base fluid used, and the desired gel characteristics.

Edible gel mixtures and edible gels can be prepared using ingredients comprising: an edible acid, an edible acid salt, a buffering system, a bulking agent, a chelating agent, a cross-linking agent, one or more flavor flavors, one or more colors, and combinations thereof. Non-limiting examples of edible acids for use in particular embodiments include citric acid, adipic acid, fumaric acid, lactic acid, malic acid, and combinations thereof. Non-limiting examples of food acid salts for use in particular embodiments include sodium salts of food acids, potassium salts of food acids, and combinations thereof. Non-limiting examples of bulking agents for use in particular embodiments include fructooligosaccharides (raftilose), isomalt, sorbitol, polydextrose, maltodextrin, and combinations thereof. Non-limiting examples of chelating agents for use in particular embodiments include calcium disodium ethylene tetraacetate, glucono delta-lactone, sodium gluconate, potassium gluconate, ethylenediaminetetraacetic acid (EDTA), and combinations thereof. Non-limiting examples of cross-linking agents useful in particular embodiments include calcium ions, magnesium ions, sodium ions, and combinations thereof.

Dental composition

In one embodiment, the present invention is a dental composition comprising rebaudioside I. In another embodiment, the present invention is a dental composition comprising a rebaudioside I containing composition. Dental compositions typically comprise an active dental substance and a base material. Rebaudioside I or compositions comprising rebaudioside I can be used as a base material to sweeten the dental composition. The dental composition may be in the form of any oral composition for use in the oral cavity, such as, for example, a mouth freshening preparation, a mouthwash, a mouth rinse, a dentifrice, a tooth polish, a dentifrice, a mouth spray, a tooth whitener, dental floss, and the like.

As referred to herein, "active dental substance" means any composition that can be used to enhance the aesthetic appearance and/or health of teeth or gums or to prevent dental caries. As referred to herein, "base material" refers to any inactive substance used as a vehicle for an active dental substance, such as any material that facilitates handling, stability, dispersibility, wettability, foaming, and/or release kinetics of an active dental substance.

Suitable active dental substances for use in embodiments of the present invention include, but are not limited to, substances that remove plaque, substances that remove food from teeth, substances that help eliminate and/or mask bad breath, substances that prevent dental caries, and substances that prevent gum disease (i.e., gums). Examples of suitable active dental substances for use in embodiments of the present invention include, but are not limited to, anticaries agents, fluorides, sodium fluoride, sodium monofluorophosphate, stannous fluoride, hydrogen peroxide, urea peroxide (i.e., urea peroxide), antibacterial agents, plaque removal agents, detergents, anticalculus agents, abrasives, baking soda, percarbonate, perborate salts of alkali and alkaline earth metals, or similar types of substances, or combinations thereof. Such components are generally considered safe (GRAS) and/or are U.S. Food and Drug Administration (FDA) approved.

According to a specific embodiment of the present invention, the active tooth substance is present in the dental composition in an amount ranging from about 50ppm to about 3000ppm of the dental composition. Typically, the active dental composition is present in the dental composition in an amount effective to at least marginally enhance the aesthetic appearance and/or health of the teeth or gums or to prevent dental caries. For example, a dental composition comprising a dentifrice may comprise an active dental substance comprising fluorine in an amount of about 850 to 1,150 ppm.

In addition to rebaudioside I or a composition comprising rebaudioside I, the dental composition may further comprise a base material. Examples of suitable substrate materials for embodiments of the present invention include, but are not limited to, water, sodium lauryl sulfate or other sulfate, wetting agents, enzymes, vitamins, herbs, calcium, flavoring agents (e.g., mint, bubble gum, cinnamon, lemon, or orange), surfactants, binders, preservatives, gelling agents, pH adjusters, peroxide activators, stabilizers, colorants, or similar types of materials, and combinations thereof.

The base material of the dental composition may optionally include other artificial or natural sweeteners, bulk sweeteners, or combinations thereof. Bulk sweeteners include both caloric and non-caloric compounds. Non-limiting examples of bulk sweeteners include sucrose, dextrose, maltose, dextrin, dried invert sugar, fructose, high fructose corn syrup, levulose, galactose, corn syrup solids, tagatose, polyols (e.g., sorbitol, mannitol, xylitol, lactitol, erythritol, and maltitol), hydrogenated starch hydrolysates, isomalt, trehalose, and mixtures thereof. Generally, the amount of bulk sweetener present in the dental composition is a wide range depending on the particular embodiment of the dental composition and the sweetness desired. One of ordinary skill in the art will readily determine the appropriate amount of bulk sweetener. In particular embodiments, the bulk sweetener is present in the dental composition in an amount in the range of about 0.1% to about 5% by weight of the dental composition.

According to a specific embodiment of the present invention, the base material is present in the dental composition in an amount ranging from about 20% to about 99% by weight of the dental composition. Typically, the base material is present in an amount effective to provide a vehicle for an active dental substance.

In a particular embodiment, a dental composition comprises rebaudioside I and an active dental substance. In another particular embodiment, a dental composition comprises a composition comprising rebaudioside I and an active dental substance. Generally, the amount of such sweeteners will vary widely depending on the nature of the particular dental composition and the sweetness level desired.

Food products include, but are not limited to, confections, condiments, chewing gum, cereals, baked goods, and dairy products.

Sweet food

In one embodiment, the invention is a rebaudioside I-containing confection. In another embodiment, the invention is a confection comprising a rebaudioside I containing composition.

As referred to herein, "confectionery" may mean candy, sugar (lillie), pastry candy or similar terms. Confections typically comprise a base component and a sweetener component. Rebaudioside I or compositions comprising rebaudioside I can be used as the sweetener component. The confectionery may be in any food form which is typically considered to be sugar rich or typically a candy. According to a particular embodiment of the invention, the confectionery may be a baked good, such as a pastry; desserts, such as yogurt, jelly, drinkable jelly, pudding, bavaria cream, custard, cake, chocolate cake, mousse, etc., confectionery products to be consumed at afternoon or after meals; freezing the food; cold confections, for example, ice cream types such as ice cream, ice milk, milk-flavored ice cream (lacto-ice), and the like (foods in which sweeteners and various other types of ingredients are added to dairy products and the resulting mixture is agitated and frozen), and frozen confections such as sherbet (sherbet), snack ice cream (dessert ice), and the like (foods in which various other types of ingredients are added a sugar-containing liquid and the resulting mixture is agitated and frozen); general confectionery such as baked confectionery or steamed confectionery such as crackers, biscuits, buns with bean jam fillings, sesame crunchy candies, sweet milk sandwich cakes (alfajor) and the like; rice cakes and snacks; a desktop product; sugar confections in general, such as chewing gums (including, for example, compositions containing a substantially water-insoluble, chewable gum base, such as chicle (chicle) or alternatives thereof, including jelutong (jetulong), guttakay (guttakay) rubber or some edible natural synthetic resin or wax), hard candies, soft candies, mints, nougats, soft toffee, fudge, taffy, swiss tablets, licorice, chocolate candy, gel candy, marshmallow, marzipan (r), fudge (divinity), marshmallow, and the like; sauces, including fruit flavored sauces, chocolate sauces, and the like; edible gel; creams, including butter cream, masa, raw butter, and the like; jams, including strawberry jam, citrus jam, and the like; and bread, including sweet bread and the like or other starch products, and combinations thereof.

As referred to herein, a "base composition" means any composition that can be a food product and provides a matrix for carrying sweetener components.

Suitable base compositions for use in embodiments of the present invention may include flour, yeast, water, salt, butter, eggs, milk powder, spirits, gelatin, nuts, chocolate, citric acid, tartaric acid, fumaric acid, natural flavors, artificial flavors, colors, polyols, sorbitol, isomalt, maltitol, lactitol, malic acid, magnesium stearate, lecithin, hydrogenated glucose syrup, glycerin, natural or synthetic gums, starch, and the like, as well as combinations thereof. Such components are generally considered safe (GRAS) and/or are U.S. Food and Drug Administration (FDA) approved. According to a particular embodiment of the invention, the base composition is present in the confection in an amount ranging from about 0.1% to about 99% by weight of the confection. Generally, the base composition is present in the confection in an amount to provide a food product.

The base material of the confection may optionally include other artificial or natural sweeteners, bulk sweeteners, or combinations thereof. Bulk sweeteners include both caloric and non-caloric compounds. Non-limiting examples of bulk sweeteners include sucrose, dextrose, maltose, dextrin, dried invert sugar, fructose, high fructose corn syrup, levulose, galactose, corn syrup solids, tagatose, polyols (e.g., sorbitol, mannitol, xylitol, lactitol, erythritol, and maltitol), hydrogenated starch hydrolysates, isomalt, trehalose, and mixtures thereof. Generally, the amount of bulk sweetener present in a confection varies widely depending on the particular embodiment of the confection and the sweetness desired. One of ordinary skill in the art will readily determine the appropriate amount of bulk sweetener.

In particular embodiments, the dessert comprises rebaudioside I or a composition comprising rebaudioside I and a base composition. Generally, the amount of rebaudioside I in the confections varies widely depending on the particular embodiment of the confection and the desired sweetness. One of ordinary skill in the art will readily determine the appropriate amount. In a particular embodiment, rebaudioside I is present in the confection in an amount ranging from about 30ppm to about 6000ppm of the confection. In another embodiment, rebaudioside I is present in the confection in an amount in the range of about 1 ppm to about 10,000ppm of the confection. In embodiments where the confection comprises hard candy, rebaudioside I is present in an amount in the range of about 150ppm to about 2250ppm of hard candy.

Flavouring composition

In one embodiment, the present invention is a condiment comprising rebaudioside I. In another embodiment, the invention is a condiment comprising a rebaudioside I containing composition. A flavoring, as used herein, is a composition used to enhance or improve the flavor of a food or beverage. Non-limiting examples of condiments include tomato sauce (ketchup); mustard; barbecue sauce; butter; a chili sauce; sour and spicy sauce; appetizing sauce; curry powder; dipping; fish sauce; horseradish sauce; chili sauce; jelly, jam, citrus jam, or preserves; mepiride; peanut butter; appetizing dishes (relish); mayonnaise; salad dressings (e.g., oil and vinegar, kaiser sauce (Caesar), French sauce (French), pasture sauce (ranch), blue cheese, Russian sauce (Russian), thousand island sauce, Italian sauce (Italian), and balsamic juice), salsa chili sauce (salsa); german pickle; soy sauce; beefsteak sauce; syrup; tower sauce; and a Worcester sauce.

The condiment base typically comprises a mixture of different ingredients, including, without limitation, a vehicle (e.g., water and vinegar); spices or seasonings (e.g., salt, pepper, garlic, mustard seed, onion, chili pepper, turmeric, and combinations thereof); fruits, vegetables, or products thereof (e.g., tomatoes or tomato-based products (pastes, purees), juices, fruit peel juice (fruit juice), and combinations thereof); oil or oil emulsions, particularly vegetable oils; thickeners (e.g., xanthan gum, food starch, other hydrocolloids, and combinations thereof); and emulsifying agents (e.g., egg yolk solids, proteins, gum arabic, carob gum, guar gum, karaya gum, tragacanth gum, carrageenan, pectin, propylene glycol esters of alginic acid, sodium carboxymethylcellulose, polysorbates, and combinations thereof). Recipes for flavor bases and methods of making flavor bases are well known to those of ordinary skill in the art.

Typically, the flavoring will also contain a caloric sweetener such as sucrose, high fructose corn syrup, molasses, honey, or brown sugar. In exemplary embodiments of the condiments provided herein, rebaudioside I or rebaudioside I-containing sweetener compositions are used instead of traditional caloric sweeteners. Thus, a flavoring composition desirably comprises rebaudioside I or a rebaudioside I containing composition and a flavoring base.

The flavoring composition optionally can include other natural and/or synthetic high-potency sweeteners, bulk sweeteners, pH modifiers (e.g., lactic acid, citric acid, phosphoric acid, hydrochloric acid, acetic acid, and combinations thereof), bulking agents, functional agents (e.g., medicaments, nutrients, or components of food or plants), flavoring agents, coloring agents, or combinations thereof.

Chewing gum composition

In one embodiment, the present invention is a chewing gum composition comprising rebaudioside I. In another embodiment, the present invention is a chewing gum composition comprising a rebaudioside I containing composition. Chewing gum compositions generally comprise a water-soluble portion and a water-insoluble chewable gum base portion. The water soluble portion, typically comprising the composition of the present invention, disperses over a period of time as part of the flavor is chewed while the insoluble gum portion remains in the mouth. The insoluble gum base generally determines whether a gum is considered a chewing gum, bubble gum or functional gum.

The insoluble gum base is typically present in the chewing gum composition in an amount ranging from about 15% to about 35% by weight of the chewing gum composition, which typically comprises a combination of elastomers, softeners (plasticizers), emulsifiers, resins, and fillers. Such components are generally considered food grade, are considered safe (GRA) and/or are U.S. Food and Drug Administration (FDA) approved.

Elastomers are the major component of the gum base, which provides rubbery, cohesive properties to the chewing gum, and may include one or more natural rubbers (e.g., smoked latex, liquid latex, or guayule); natural gums (e.g., jelutong, peliluo (perillo), coumarone, massaranduba balata, massaranduba chocolate, manicure (nispero), rosindinha (rosindinha), chicle, and latex margarine (gutta hang kang)); or synthetic elastomers (e.g., butadiene-styrene copolymers, isobutylene-isoprene copolymers, polybutadiene, polyisobutylene, and vinyl polymeric elastomers). In a particular embodiment, the elastomer is present in the gum base in an amount ranging from about 3% to about 50% by weight of the gum base.

The resin is used to change the firmness of the gum base and to help soften the elastomeric component of the gum base. Non-limiting examples of suitable resins include rosin esters, terpene resins (e.g., terpene resins from alpha-pinene, beta-pinene, and/or d-limonene), polyvinyl acetate, polyvinyl alcohol, ethylene vinyl acetate, and vinyl acetate-vinyl laurate copolymers. Non-limiting examples of rosin esters include glycerol esters of partially hydrogenated rosin, glycerol esters of polymerized rosin, glycerol esters of partially dimerized rosin, glycerol esters of rosin, pentaerythritol esters of partially hydrogenated rosin, methyl esters of rosin, or methyl esters of partially hydrogenated rosin. In a particular embodiment, the resin is present in the gum base in an amount ranging from about 5% to about 75% by weight of the gum base.

Softeners, also known as plasticizers, are used to modify the ease of chewing and/or the mouthfeel of the chewing gum composition. Typically, emollients include oils, fats, waxes, and emulsifiers. Non-limiting examples of oils and fats include tallow, hydrogenated tallow, large hydrogenated or partially hydrogenated vegetable oils (such as soybean oil, rapeseed oil, cottonseed oil, sunflower oil, palm oil, coconut oil, corn oil, safflower oil, or palm kernel oil)), cocoa butter, glycerol monostearate, glycerol triacetate, glycerol rosinate, lecithin (leithin), monoglycerides, diglycerides, triglyceride acetylated monoglycerides, and free fatty acids. Non-limiting examples of waxes include polypropylene/polyethylene/fischer-Tropsch wax, paraffin wax, and microcrystalline and natural waxes (e.g., candelilla, beeswax, and carnauba). Microcrystalline waxes, particularly those with high crystallinity and high melting point, may also be considered thickeners or structure modifiers. In a particular embodiment, the softeners are present in the gum base in an amount in the range of about 0.5% to about 25% by weight of the gum base.

Emulsifiers are used to form a homogeneous dispersion of the insoluble and soluble phases of the chewing gum composition and also have plasticizing properties. Suitable emulsifiers include Glycerol Monostearate (GMS), lecithin (phosphatidylcholine), polyglycerol polyricinoleic acid (PPGR), mono-and diglycerides of fatty acids, glycerol distearate, triacetin, acetylated monoglycerides, triacetin, and magnesium stearate. In a particular embodiment, the emulsifiers are present in the gum base in an amount ranging from about 2% to about 30% by weight of the gum base.

The chewing gum composition may also include adjuvants or fillers in the gum base and/or soluble portion of the chewing gum composition. Suitable adjuvants and fillers include lecithin, inulin, polydextrose, calcium carbonate, magnesium silicate, limestone powder, aluminum hydroxide, aluminum silicate, talc, clay, alumina, titanium dioxide, and calcium phosphate. In particular embodiments, lecithin may be used as an inert filler to reduce the viscosity of the chewing gum composition. In other embodiments, lactic acid copolymers, proteins (e.g., gluten and/or zein), and/or guar gum may be used to form a chewing gum that is more readily biodegradable. These adjuvants or fillers are typically present in the gum base in an amount up to about 20% by weight of the gum base. Other optional ingredients include colorants, whitening agents, preservatives, and flavor seasonings.

In particular embodiments of the chewing gum composition, the gum base comprises about 5% to about 95% by weight of the chewing gum composition, more desirably about 15% to about 50% by weight of the chewing gum composition and even more desirably from about 20% to about 30% by weight of the chewing gum composition.

The soluble portion of the chewing gum composition may optionally include other artificial or natural sweeteners, bulk sweeteners, softeners, emulsifiers, flavoring agents, colorants, adjuvants, fillers, functional agents (e.g., pharmaceuticals or nutrients), or combinations thereof. Examples of suitable softeners and emulsifiers are described above.

Bulk sweeteners include both caloric and non-caloric compounds. Non-limiting examples of bulk sweeteners include sucrose, dextrose, maltose, dextrin, dried invert sugar, fructose, high fructose corn syrup, levulose, galactose, corn syrup solids, tagatose, polyols (e.g., sorbitol, mannitol, xylitol, lactitol, erythritol, and maltitol), hydrogenated starch hydrolysates, isomalt, trehalose, and mixtures thereof. In particular embodiments, the bulk sweetener is present in the chewing gum composition in an amount in the range of about 1% to about 75% by weight of the chewing gum composition.

Flavoring agents may be used in the insoluble gum base or soluble portion of the chewing gum composition. Such flavoring agents may be natural flavors or artificial flavors. In one embodiment, the flavoring agent comprises essential oils, such as plant or fruit derived oils, peppermint oil, spearmint oil, other mint oils, clove oil, cinnamon oil, oil of wintergreen, bay oil, thyme oil, cedar leaf oil, oil of nutmeg, oil of allspice, oil of sage, oil of mace, and oil of almond. In another embodiment, the flavoring agent comprises a plant extract or fruit essence, such as apple, banana, watermelon, pear, peach, grape, strawberry, raspberry, cherry, plum, pineapple, apricot, and mixtures thereof. In yet another embodiment, the flavoring agent comprises citrus flavoring agents, such as extracts, essences, or oils of lemon, lime, orange, tangerine, grapefruit, citron, or kumquat.

In a particular embodiment, a chewing gum composition comprises rebaudioside I or a composition comprising rebaudioside I and gum base. In a particular embodiment, rebaudioside I is present in the chewing gum composition in an amount in the range of about 1ppm to about 10,000ppm of the chewing gum composition.

Cereal compositions

In one embodiment, the present invention is a grain composition comprising rebaudioside I. In another embodiment, the invention is a grain composition comprising a rebaudioside I containing composition. The cereal composition is typically consumed as a staple food or as a snack. Non-limiting examples of grain compositions for use in particular embodiments include ready-to-eat grains as well as hot grains. Ready-to-eat cereals are cereals that can be eaten without further processing (i.e. cooking) by the consumer. Examples of ready-to-eat cereals include breakfast cereals and snack bars. Breakfast cereals are typically processed to produce a shredded, flaked, expanded or extruded form. Breakfast cereals are usually consumed chilled and usually mixed with milk and/or fruit. Snack bars include, for example, energy bars, rice cakes, granola bars, and nutritional bars. The hot cereal is usually cooked with milk or water before being eaten. Non-limiting examples of hot cereals include coarse oat flour, porridge, corn porridge, rice, and oatmeal.

The cereal composition typically comprises at least one cereal ingredient. As used herein, the term "grain component" refers to materials such as whole or partial grains, whole or partial seeds, and whole or partial grasses. Non-limiting examples of grain components for use in particular embodiments include corn, wheat, rice, barley, bran endosperm (bran endosperms), milled dried wheat (bulgur), sorghum, millet, oats, rye, triticale, buckwheat, fonio (fonio), quinoa, beans, soybeans, amaranth, teff, spelt, and kavani (kaniwa).

In a particular embodiment, the grain composition comprises rebaudioside I or a composition comprising rebaudioside I and at least one grain component. Rebaudioside I or compositions comprising rebaudioside I can be added to the grain composition in a variety of ways, for example, as a coating, as a cream, as a glaze, or as a base blend (i.e., added to the grain formulation as an ingredient prior to preparing the final grain product).

Thus, in a particular embodiment, rebaudioside I or a composition comprising rebaudioside I is added to the grain composition as a base blend. In one embodiment, rebaudioside I or a composition comprising rebaudioside I is blended with a hot cereal prior to cooking to provide a sweetened hot cereal product. In another embodiment, rebaudioside I or a composition comprising rebaudioside I is blended with the cereal matrix prior to pressing the cereal.

In another particular embodiment, rebaudioside I or a composition comprising rebaudioside I is added to the grain composition as a coating, such as, for example, by combining rebaudioside I or a composition comprising rebaudioside I with a food grade oil and applying the mixture to the grain. In a different embodiment, rebaudioside I or a composition comprising rebaudioside I and a food grade oil can be applied separately to the grain by first applying the oil or sweetener. Non-limiting examples of food grade oils for use in particular embodiments include vegetable oils, such as corn oil, soybean oil, cottonseed oil, peanut oil, coconut oil, rapeseed oil, olive oil, sesame seed oil, palm kernel oil, and mixtures thereof. In another embodiment, food grade fats may be used in place of the oils, provided that the fat is melted prior to application to the grain.

In another embodiment, rebaudioside I or a composition comprising rebaudioside I is added to the grain composition as a glaze. Non-limiting examples of glazing agents for use in particular embodiments include corn syrup, honey, syrup and honey syrup solids, maple syrup and maple syrup solids, sucrose, isomalt, polydextrose, polyols, hydrogenated starch hydrolysates, aqueous solutions thereof, and mixtures thereof. In another such embodiment, rebaudioside I or a composition comprising rebaudioside I is added as a glaze by combining with a polish and an edible grade oil or fat and applying the mixture to the grain. In another embodiment, a gum system, such as, for example, gum arabic, carboxymethyl cellulose, or algin, may be added to the glaze to provide structural support. In addition, the glaze may also contain a colorant, and may also contain a fragrance.

In another embodiment, rebaudioside I or a composition comprising rebaudioside I is added to the grain composition as a cream. In one such embodiment, rebaudioside I or a composition comprising rebaudioside I is combined with water and a frosting agent and then applied to the grain. Non-limiting examples of frosting agents useful in particular embodiments include maltodextrin, sucrose, starch, polyols, and mixtures thereof. The cream may also comprise a food grade oil, food grade fat, colorant, and/or fragrance.

Generally, the amount of rebaudioside I in the grain composition varies widely depending on the particular type of grain composition and its desired sweetness. One of ordinary skill in the art can readily determine the appropriate amount of sweetener to add to the cereal composition. In a particular embodiment, rebaudioside I is present in the grain composition in an amount ranging from about 0.02 wt% to about 1.5 wt% of the grain composition, and the at least one additive is present in the grain composition in an amount ranging from about 1 wt% to about 5 wt% of the grain composition.

Baked food

In one embodiment, the present invention is a baked good comprising rebaudioside I. In another embodiment, the present invention is a baked good comprising a rebaudioside I containing composition. As used herein, baked goods include ready-to-eat and all ready-to-bake products, flours and mixtures that need to be prepared prior to serving. Non-limiting examples of baked goods include cakes, crackers, cookies, chocolates, muffins, rolls, bagels, donuts, tarts, pastries, croissants, cookies, bread products, and buns.

Preferred baked goods according to embodiments of the present invention may be classified into three groups: bread-type doughs (e.g., white bread, flavored bread (variegated), soft bread, hard rolls, bagels, pizza dough, and mexican wafers), sweet doughs (e.g., danish crusts, croissants, crackers, multi-layers, pie crusts, cookies, and cookies), and batters (e.g., cakes such as sponge cakes, pound cakes, devil cakes, cheesecakes, and sandwich cakes, doughnuts or other yeast-leavened cakes, chocolate cakes, and muffins). Dough is typically characterized as flour-based, whereas batter is more water-based.

Baked goods according to particular embodiments of the invention typically comprise a combination of sweetener, water and fat. Baked goods prepared according to many embodiments of the present invention also contain flour to prepare a dough or batter. The term "dough" as used herein is a mixture of flour and other ingredients that is sufficiently hard to knead or roll. The term "batter" as used herein consists of flour, liquid such as milk or water, and other ingredients and is thin enough to pour or drip from a spoon. Desirably, according to particular embodiments of the present invention, the flour is present in the baked good in an amount ranging from about 15% to about 60% on a dry weight basis, more desirably from about 23% to about 48% on a dry weight basis.

The type of flour may be selected based on the desired product. Typically, the flour comprises edible non-toxic flour commonly used in baked goods. According to particular embodiments, the flour may be a bleached toast flour, a universal flour, or an unbleached flour. In other embodiments, flours treated in other ways may also be used. For example, in particular embodiments, the flour may be enriched with additional vitamins, minerals, or proteins. Non-limiting examples of flours suitable for use in embodiments of the invention include wheat flour, corn flour, whole grains, portions of whole grains (wheat, bran, and oatmeal), and combinations thereof. In particular embodiments, starch or starch-containing materials may also be used as the flour. Common food starches are typically derived from potato, corn, wheat, barley, oat, tapioca, arrowroot, and sago. In particular embodiments of the present invention, modified and pregelatinized starches may be used.

The type of fat or oil used in particular embodiments of the present invention may include any edible fat, oil, or combination thereof suitable for baking. Non-limiting examples of fats suitable for use in particular embodiments of the present invention include vegetable oils, tallow, lard, marine oils, and combinations thereof. According to particular embodiments, these fats may be fractionated, partially hydrogenated, and/or fortified. In another embodiment, the fat desirably comprises reduced, low-calorie, or non-digestible fat, fat substitute, or synthetic fat. In another embodiment, shortening, fat, or a mixture of hard and soft fats may also be used. In particular embodiments, the shortening may be derived in large part from triglycerides derived from vegetable sources (e.g., cottonseed oil, soybean oil, peanut oil, linseed oil, sesame oil, palm kernel oil, rapeseed oil, safflower oil, coconut oil, corn oil, sunflower oil, and mixtures thereof). In particular embodiments, synthetic or natural triglycerides of fatty acids having chain lengths of from 8 to 24 carbon atoms may also be used. Desirably, according to a specific embodiment of the present invention, the fat is present in the baked good in an amount ranging from about 2% to about 35% by weight on a dry basis, more desirably from about 3% to about 29% by weight on a dry basis.

Baked goods according to particular embodiments of the invention also contain water in an amount sufficient to provide the desired consistency, thereby enabling proper shaping, machining and cutting of the baked goods before or after cooking. The total moisture content of the baked good includes any water added directly to the baked good as well as water present in the separately added ingredients (e.g., flour, which typically contains about 12% to about 14% moisture by weight). Desirably, according to a particular embodiment of the present invention, the water is present in the baked good in an amount up to about 25% by weight of the baked good.

Baked goods according to embodiments of the invention may also contain a variety of additional common ingredients such as leaveners, flavors, colors, milk by-products, eggs, egg by-products, cocoa, vanilla or other flavorings, as well as inclusions such as nuts, raisins, cherries, apples, apricots, peaches, other fruits, citrus peels, preservatives, coconuts, flavor chips (such as chocolate chips, butterscotch chips and caramel chips), and combinations thereof. In particular embodiments, the baked good may further comprise an emulsifier, such as lecithin and monoglycerides.

According to embodiments of the present invention, the leavening agent may comprise a chemical leavening agent or a yeast leavening agent. Non-limiting examples of chemical leavening agents suitable for use in particular embodiments of the present invention include baking soda (e.g., sodium bicarbonate, potassium bicarbonate, or aluminum bicarbonate), fermentation acids (e.g., sodium aluminum phosphate, monocalcium phosphate, or dicalcium phosphate), and combinations thereof.

According to another embodiment of the invention, the cocoa may comprise natural or "base-treated" chocolate, the majority of which fat or cocoa butter is expressed or removed by solvent extraction, extrusion or other means. In a particular example, it may be desirable to reduce the amount of fat in a baked food product comprising chocolate because of the additional fat present in cocoa butter. In particular embodiments, a greater amount of chocolate may need to be added than cocoa in order to provide an equivalent amount of flavor and color.

Baked goods typically also contain caloric sweeteners such as sucrose, high fructose corn syrup, erythritol, molasses, honey, or brown sugar. In exemplary embodiments of the baked goods provided herein, the caloric sweetener is replaced partially or completely with rebaudioside I or a composition comprising rebaudioside I. Thus, in one embodiment, a baked good comprises rebaudioside I or a composition comprising rebaudioside I in combination with fat, water, and optionally ground flour. In particular embodiments, the baked good can optionally include other natural and/or synthetic high-potency sweeteners and/or bulk sweeteners.

Dairy product

In one embodiment, the consumable of the present invention is a dairy product comprising rebaudioside I. In another embodiment, the consumable of the present invention is a milk product comprising a rebaudioside I containing composition. The dairy products and methods for preparing dairy products suitable for use in the present invention are well known to those of ordinary skill in the art. As used herein, a dairy product includes milk or a food product produced from milk. Non-limiting examples of dairy products suitable for use in embodiments of the invention include milk, cream, sour cream, french creme, buttermilk, fermented buttermilk, milk powder, condensed milk, evaporated milk, butter, cheese, cottage cheese, cream cheese, yogurt, ice cream, soft savory jelly, frozen yogurt, spaghetti (gelato), margarine (vla), healthy yogurt (piima), yogurt

Karok (kajmak), kefir (kephir), willi (viii), kumis (kumis), argy yogurt (airag), ice milk, casein, salt yogurt (ayran), hindu (lassi), korean condensed milk (khoa), or combinations thereof.

Milk is a fluid secreted by the mammary glands of female mammals that is used to nurture their litters. The female capacity to produce milk is one of the defined mammalian traits and provides the primary source of nutrients for newborns before they can digest more diverse foods. In particular embodiments of the invention, the dairy products are raw milk derived from cattle, goats, sheep, horses, donkeys, camels, buffalo, yak, reindeer, moose, or humans.

In a particular embodiment of the invention, processing of a dairy product from raw milk typically comprises the steps of pasteurization, creaming and homogenization. Although raw milk can be consumed without pasteurization, it is typically pasteurized to destroy harmful microorganisms, such as bacteria, viruses, protozoa, molds, and yeasts. Pasteurization typically involves heating the milk to an elevated temperature for a short period of time to substantially reduce the number of microorganisms, thereby reducing the risk of disease.

Creaming is traditionally after a pasteurization step and involves the separation of milk into a higher fat milk layer and a lower fat milk layer. The milk will separate into a milk layer and a cream layer after being left for twelve to twenty-four hours. The cream rises to the top of the milk layer and can be skimmed off and used as a separate dairy product. Alternatively, centrifugation can be used to separate cream from milk. The remaining milk is classified according to the fat content of the milk, non-limiting examples of which include whole milk, 2% fat milk, 1% fat milk, and skim milk.

After the desired amount of fat is removed from the milk by creaming, the milk is often homogenized. Homogenization prevents cream from separating from milk and generally involves pumping milk through a small diameter tube at high pressure in order to break up fat globules in the milk. Pasteurization, creaming, and homogenization of milk are common but not necessary for the production of consumable dairy products. Thus, suitable dairy products for use in embodiments of the present invention may be processed without processing steps, in a single processing step, or in a combination of processing steps described herein. Suitable dairy products for use in embodiments of the invention may be subjected to processing steps other than those described herein.

Particular embodiments of the present invention include dairy products produced from milk by additional processing steps. As mentioned above, cream may be skimmed from the top of the milk or separated from the milk using a machine centrifuge. In a particular embodiment, the dairy product comprises sour cream, which is a dairy product rich in fat obtained using a bacterial culture fermented cream. The bacteria produce lactic acid during fermentation, which turns the cream sour and thick. In another embodiment, the dairy product comprises french whipped cream, which is a multi-fat cream that is slightly acidified with bacterial culture in a manner similar to sour cream. French whipped cream is generally not as thick or as sour cream. In another embodiment, the dairy product comprises fermented buttermilk. Fermented buttermilk is obtained by adding bacteria to milk. Wherein the resulting fermentation of the bacterial culture to convert lactose to lactic acid produces a fermented buttermilk that is sour in taste. Although it is produced in a different way, fermented buttermilk is generally similar to traditional buttermilk, which is a by-product of butter manufacture.

According to other specific embodiments of the present invention, the dairy product comprises milk powder, condensed milk, evaporated milk or a combination thereof. Milk powder, condensed milk and evaporated milk are typically produced by removing water from milk. In a particular embodiment, the dairy product comprises a milk powder containing dry milk solids having a low moisture content. In another embodiment, the dairy product comprises condensed milk. Condensed milk typically contains milk with a reduced moisture content and added sweeteners, resulting in a thick sweetened product with a long shelf life. In another embodiment, the dairy product comprises evaporated milk. Evaporated milk typically comprises fresh, homogenized milk from which about 60% of the water has been removed, which has been cooled, fortified with additives such as vitamins and stabilizers, packaged, and terminally sterilized. According to another specific embodiment of the present invention, a dairy product comprises a dry creamer and rebaudioside I or a composition comprising rebaudioside I.

In another particular embodiment, the dairy product provided herein comprises butter. Butter is typically prepared by stirring fresh or fermented cream or milk. Butter typically comprises milk fat around small droplets containing mostly water and milk proteins. The stirring process damages the membrane around the cream globules, allowing the cream to bind and separate from the rest of the cream. In another embodiment, the dairy product comprises buttermilk, which is a sour liquid retained after butter is produced from whole milk by a blending process.

In another embodiment, the dairy product comprises cheese, which is a solid food product produced by coagulating milk using rennet or a combination of rennet substitutes and acidification. Rennet is a natural enzyme complex produced in the stomach of a mammal that digests milk, which is used in cheese making to coagulate milk so that it separates into a solid called curd and a liquid called whey. Generally, chymosin is obtained from the stomach of young ruminants such as calves; however, alternative sources of rennet include some plants, microbial organisms, and genetically engineered bacteria, fungi, or yeast. Furthermore, the milk may be coagulated by adding acid, such as citric acid. Typically, a combination of rennet and/or acidification is used to coagulate the milk. After separating the milk into curds and whey, some cheeses are made by simply draining, salting, and packaging the curds. However, for most cheeses, more processing is required. Many different methods can be used to produce hundreds of available types of cheese. The process includes heating the cheese, cutting it into pieces for draining (drain), salting, stretching, forming cheddar cheese, washing, molding, ripening, and ripening. Some cheeses, such as blue cheese, have additional bacteria or molds introduced into them before or during ripening, thereby imparting flavor and aroma to the final product. White soft cheese is a cheese curd product with a desirable flavor that is drained but not squeezed so that some whey is retained. The curd is typically washed to remove acidity. Cream cheese is a soft, moderately flavored white cheese with a high fat content that is produced by adding cream to milk and then coagulating it to form an enriched curd. Alternatively, cream cheese may be made from skim milk, where cream is added to the curd. It is to be understood that cheese as used herein includes all solid food products produced by coagulating milk.

In another embodiment of the invention, the dairy product comprises yogurt. Yogurt is typically produced by bacterial fermentation of milk. The fermentation of lactose produces lactic acid, which acts on the proteins in the milk to produce a gel-like texture and sour taste. In particularly desirable embodiments, the yogurt may be sweetened and/or flavored with a sweetener. Non-limiting examples of flavoring agents include, but are not limited to, fruit (e.g., peach, strawberry, banana), vanilla, and chocolate. As used herein, yogurt also includes yogurt varieties of different consistencies and viscosities, such as darcy yogurt (dahi), darchi (dadifh) or dardadiah, concentrated yogurt (labneh) or mediterranean yogurt (labaneh), bulgarian yogurt (bulgarian), kefir (kefir), and liya yogurt (matsoni). In another embodiment, the dairy product comprises a yogurt based beverage, also known as drinkable yogurt or a yogurt smoothie. In particularly desirable embodiments, the yogurt-based beverage may contain sweeteners, flavoring agents, other ingredients, or combinations thereof.

In particular embodiments of the present invention, other dairy products than those described herein may be used. Such dairy products are well known to those of ordinary skill in the art, non-limiting examples of which include milk, milk and juice, coffee, tea, miso (vla), healthy yogurt, yogurt (filmjolk), karjmak (kajmak), kefir (kephir), willi (viii), margaris (kumis), illige (airag), ice milk, casein, salt yogurt (ayran), indian milkshakes (lassi), and korean condensed milk (khoa).

According to a particular embodiment of the invention, the dairy composition may also comprise other additives. Non-limiting examples of suitable additives include sweetening agents and flavoring agents, such as chocolate, strawberry, and banana. Particular embodiments of the dairy compositions provided herein can also include additional nutrient supplements such as vitamins (e.g., vitamin D) and minerals (e.g., calcium) to improve the nutrient composition of milk.

In a particularly desirable embodiment, the dairy composition comprises rebaudioside I or a rebaudioside I containing composition in combination with a dairy product. In a particular embodiment, rebaudioside I is present in the dairy composition in an amount ranging from about 200% to about 20,000% by weight of the dairy composition.

Rebaudioside I or compositions comprising rebaudioside I are also suitable for processed agricultural products, livestock products, or seafood; processed meat products such as sausages and the like; retort food, pickled product, jam boiled in soy sauce, delicacies, and side dish; soup; snacks, such as potato chips, sweet cookies, etc.; chopped filler, leaves, stems, homogenized roasted leaves, and animal feed.

Tabletop sweetener compositions

In one embodiment, the invention is a tabletop sweetener comprising rebaudioside I. The tabletop composition can further comprise at least one bulking agent, additive, anti-caking agent, functional ingredient, or combination thereof.

Suitable "bulking agents" include, but are not limited to, maltodextrin (10DE, 18DE, or 5DE), corn syrup solids (20DE or 36DE), sucrose, fructose, glucose, invert sugar, sorbitol, xylose, ribulose, mannose, xylitol, mannitol, galactitol, erythritol, maltitol, lactitol, isomalt, maltose, tagatose, lactose, inulin, glycerol, propylene glycol, polyols, polydextrose, fructooligosaccharides, cellulose and cellulose derivatives, and the like, and mixtures thereof. In addition, according to still other embodiments of the present invention, granulated sugar (sucrose) or other caloric sweeteners such as crystalline fructose, other carbohydrates or sugar alcohols may be used as a bulking agent because they provide good content uniformity without adding a large number of calories.

As used herein, the phrases "anti-caking agent" and "glidant" refer to any composition that contributes to content uniformity and uniform dissolution. Non-limiting examples of anti-caking agents according to specific embodiments include tartaric acid, calcium silicate, silicon dioxide, microcrystalline cellulose (Avicel, FMC BioPolymer, philiadelphia, Pennsylvania) from FMC BioPolymer corporation, Philadelphia, Pennsylvania), and tricalcium phosphate. In one embodiment, the anti-caking agent is present in the tabletop sweetener composition in an amount from about 0.001% to about 3% by weight of the tabletop sweetener composition.

These tabletop sweetener compositions can be packaged in any form known in the art. Non-limiting forms include, but are not limited to, powder forms, granular forms, packets, tablets, sachets, pellets, cubes, solids, and liquids.

In one embodiment, the tabletop sweetener composition is a single use (portion control) package containing a dry blend. Dry blend formulations may generally include powders or granules. Although the tabletop sweetener composition may be in one packet of any size, a non-limiting example of a conventional portion control tabletop sweetener packet is about 2.5 x 1.5 inches and retains about 1 gram of sweetener composition having a sweetness equivalent to 2 teaspoons of granulated sugar (about 8 g). The amount of rebaudioside I in a tabletop sweetener formulation of a dry blend can vary. In a particular embodiment, a tabletop sweetener formulation of a dry blend can contain rebaudioside I in an amount from about 1% (w/w) to about 10% (w/w) of the tabletop sweetener composition.

Examples of solid tabletop sweeteners include cubes and tablets. One non-limiting example of a conventional cube is a standard cube of size equivalent to granulated sugar, which is about 2.2 × 2.2 × 2.2cm ³And weighs about 8 g. In one embodiment, the solid tabletop sweetener is in the form of a tablet or any other form known to those skilled in the art.

The tabletop sweetener compositions can also be embodied in liquid form, wherein rebaudioside I is combined with a liquid carrier. Suitable non-limiting examples of carrier agents for liquid tabletop sweeteners include water, alcohols, polyols, glyceryl or citric acid groups dissolved in water, and mixtures thereof. The sweetness equivalent of any of the forms of tabletop sweetener compositions described herein or known in the art may be varied to achieve a desired sweetness profile. For example, the tabletop sweetener composition may comprise a sweetness comparable to that of a comparable amount of standard sugar. In another embodiment, the tabletop sweetener composition may comprise up to 100 times the sweetness intensity of a comparable amount of sugar. In another example, the tabletop sweetener composition may comprise up to 90 times, 80 times, 70 times, 60 times, 50 times, 40 times, 30 times, 20 times, 10 times, 9 times, 8 times, 7 times, 6 times, 5 times, 4 times, 3 times, and 2 times the sweetness intensity of a comparable amount of sugar.

Beverage and beverage product

In one embodiment, the invention is a beverage or beverage product comprising rebaudioside I. In another embodiment, the invention is a beverage or beverage comprising a rebaudioside I containing composition (e.g., a sweetener composition).

As used herein, a "beverage product" is a ready-to-drink beverage, a beverage concentrate, a beverage syrup, or a beverage powder-brewed beverage. Suitable ready-to-drink beverages include carbonated beverages and non-carbonated beverages. Carbonated beverages include, but are not limited to, enhanced sparkling beverages, colas, lemon-lime flavored sparkling beverages, orange flavored sparkling beverages, grape flavored sparkling beverages, strawberry flavored sparkling beverages, pineapple flavored sparkling beverages, ginger ale, sparkling waters, and root sparkling waters. Non-carbonated beverages include, but are not limited to, fruit juice, fruit flavored juice, fruit juice drinks, nectar, vegetable juice, vegetable flavored juice, sports drinks, energy drinks, enhanced water with vitamins, near water drinks (e.g., water with natural or synthetic flavoring agents), coconut water, tea-type drinks (e.g., dark tea, green tea, black tea, oolong tea), coffee, cocoa drinks, drinks containing milk components (e.g., milk drinks, coffee containing milk components, euro coffee (cafeau lait), milk tea, fruit milk drinks), beverages containing grain extracts, smoothies, and combinations thereof.

Beverage concentrates and beverage syrups are prepared with an initial volume of liquid base (e.g., water) and the desired beverage ingredients. A full strength beverage is then prepared by adding an additional volume of water. Beverage powder brewed beverages are prepared by dry mixing all beverage ingredients in the absence of a liquid base. A full strength beverage (full strength beverage) is then prepared by adding the entire volume of water.

The beverage contains a liquid base, i.e. the base in which the ingredients, including the composition of the invention, are dissolved. In one embodiment, the beverage comprises beverage quality water as the liquid base, such as, for example, deionized water, distilled water, reverse osmosis water, carbonated water, purified water, demineralized water, and combinations thereof, may be used. Additional suitable liquid substrates include, but are not limited to, phosphoric acid, phosphate buffer, citric acid, citrate buffer, and carbon treated water.

In one embodiment, the consumable of the present invention is a beverage comprising rebaudioside I.

In another embodiment, the beverage contains a composition comprising rebaudioside I.

In yet another embodiment, the invention is a beverage product comprising rebaudioside I.

In another embodiment, the invention is a beverage product containing a composition comprising rebaudioside I.

The concentration of rebaudioside I in the beverage or beverage product can be above, at, or below its threshold sweetness or identifying concentration.

In a particular embodiment, the concentration of rebaudioside I in the beverage or beverage product is above its threshold sweetness or flavor recognition concentration. In one embodiment, the concentration of rebaudioside I is at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% or greater above its threshold sweetness or flavor recognition concentration.

In another particular embodiment, the concentration of rebaudioside I in the beverage or beverage product is at or about the threshold sweetness or flavor recognition concentration of rebaudioside I.

In yet another particular embodiment, the concentration of rebaudioside I in the beverage or beverage product is less than the threshold sweetness or flavor recognition concentration of rebaudioside I. In one embodiment, the concentration of rebaudioside I is at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% or greater below its threshold sweetness or flavor recognition concentration.

In one embodiment, the present invention is a beverage or beverage product containing rebaudioside I in an amount ranging from about 1ppm to about 10,000ppm, e.g., from about 5ppm to about 10,000ppm, from about 10ppm to about 10,000ppm, from about 15ppm to about 10,000ppm, from about 20ppm to about 10,000ppm, or from about 25ppm to about 10,000 ppm. In another embodiment, rebaudioside I is present in the beverage in an amount ranging from about 100ppm to about 600 ppm. In other embodiments, rebaudioside I is present in the beverage in an amount ranging from about 100 to about 200ppm, from about 100ppm to about 300ppm, from about 100ppm to about 400ppm, or from about 100ppm to about 500 ppm. In another embodiment, rebaudioside I is present in the beverage or beverage product in an amount ranging from about 300ppm to about 700ppm, such as, for example, from about 400ppm to about 600 ppm. In another embodiment, rebaudioside I is present in the beverage or beverage product in an amount ranging from about 200ppm to about 400ppm, e.g., about 200 to about 250ppm, about 250ppm to about 300ppm, about 300ppm to about 350ppm, or about 350 ppm. In another embodiment, rebaudioside I is present in the beverage or beverage product in an amount ranging from about 450ppm to about 650ppm, e.g., about 450ppm to about 500ppm, about 500ppm to about 550ppm, about 550ppm to about 600ppm or about 600 to about 650 ppm.

In one exemplary embodiment, the beverage of the present invention is a beverage comprising rebaudioside I in an amount from about 200 to about 300ppm, more particularly about 200ppm, about 225, about 250ppm, about 275ppm, or about 300 ppm.

In one exemplary embodiment, the beverage of the present invention is rebaudioside I in an amount from about 500 to about 600ppm, more particularly about 500ppm, about 525ppm, about 550ppm, about 575ppm, or about 600 ppm.

The beverage may further comprise at least one additional sweetener. Any sweetener detailed herein can be used, including natural sweeteners, non-natural sweeteners, or synthetic sweeteners. These can be added to the beverage before, simultaneously with, or after rebaudioside I.

In one embodiment, the beverage comprises a concentration of carbohydrate sweetener from about 100ppm to about 140,000 ppm. The synthetic sweetener may be present in the beverage at a concentration of from about 0.3ppm to about 3,500 ppm. The natural high-potency sweetener may be present in the beverage at a concentration of from about 0.1ppm to about 3,000 ppm.

The beverage may further comprise additives including, but not limited to, carbohydrates, polyols, amino acids and their corresponding salts, polyamino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic salts (including organic acid salts and organic base salts), inorganic salts, bitter compounds, caffeine, flavoring agents and flavor ingredients, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, weighting agents, juices, dairy products, grains and other plant extracts, flavonoids, alcohols, polymers, and combinations thereof. Any suitable additive described herein may be used.

In one embodiment, the polyol may be present in the beverage at a concentration of from about 100ppm to about 250,000ppm, such as, for example, from about 5,000ppm to about 40,000 ppm.

In another embodiment, the amino acid may be present in the beverage at a concentration of from about 10ppm to about 50,000ppm, such as, for example, from about 1,000ppm to about 10,000ppm, from about 2,500ppm to about 5,000ppm, or from about 250ppm to about 7,500 ppm.

In yet another embodiment, the nucleotide may be present in the beverage at a concentration of from about 5ppm to about 1,000 ppm.

In another embodiment, the organic acid additive may be present in the beverage at a concentration of from about 10ppm to about 5,000 ppm.

In yet another embodiment, the mineral acid additive may be present in the beverage at a concentration of from about 25ppm to about 25,000 ppm.

In another embodiment, the bitter compound may be present in the beverage at a concentration of from about 25ppm to about 25,000 ppm.

In another embodiment, the flavoring agent may be present in the beverage at a concentration of from about 0.1ppm to about 4,000 ppm.

In yet another embodiment, the polymer may be present in the beverage at a concentration of from about 30ppm to about 2,000 ppm.

In another embodiment, the protein hydrolysate can be present in the beverage at a concentration of from about 200ppm to about 50,000 ppm.

In another embodiment, the surfactant additive may be present in the beverage at a concentration of from about 30ppm to about 2,000 ppm.

In yet another embodiment, the flavonoid additive may be present in the beverage at a concentration of from about 0.1ppm to about 1,000 ppm.

In another embodiment, the alcohol additive may be present in the beverage at a concentration of from about 625ppm to about 10,000 ppm.

In yet another embodiment, the astringency additive can be present in the beverage at a concentration of from about 10ppm to about 5,000 ppm.

The beverage may further contain one or more functional ingredients as detailed above. Functional ingredients include, but are not limited to, vitamins, minerals, antioxidants, preservatives, glucosamine, polyphenols, and combinations thereof. Any suitable functional ingredient described herein may be used.

It is contemplated that the pH of a consumable (such as, for example, a beverage) does not substantially or adversely affect the taste of the sweetener. One non-limiting example of the pH range of the beverage can be from about 1.8 to about 10. Another example includes a pH range from about 2 to about 5. In a particular embodiment, the pH of the beverage may be from about 2.5 to about 4.2. Those skilled in the art will appreciate that the pH of the beverage may vary based on the type of beverage. For example, the dairy beverage may have a pH greater than 4.2.

The titratable acidity of a beverage comprising rebaudioside I, for example, can range from about 0.01% to about 1.0% by weight of the beverage.

In one embodiment, the foamed beverage product has an acidity of from about 0.01% to about 1.0% by weight of the beverage, such as, for example, from about 0.05% to about 0.25% by weight of the beverage.

Carbonation of a foamed beverage product has from 0 to about 2% (w/w) carbon dioxide or its equivalent, for example from about 0.1% to about 1.0% (w/w).

The temperature of the beverage may range, for example, from about 4 ℃ to about 100 ℃, such as, for example, from about 4 ℃ to about 25 ℃.

The beverage may be a full calorie beverage having up to about 120 calories per 8 ounce serving.

The beverage may be a mid-calorie beverage having up to about 60 calories per 8 ounce serving.

The beverage may be a low calorie beverage having up to about 40 calories per 8 ounce serving.

The beverage may be a zero calorie beverage having less than about 5 calories per 8 ounce serving.

Application method

The compounds and compositions of the present invention may be used to impart sweetness or to enhance the flavor or sweetness of a consumable or other composition.

In another aspect, the present invention is a method for preparing a consumable product comprising (I) providing a consumable base and (ii) adding rebaudioside I to the consumable base to provide a consumable product.

In a particular embodiment, the present invention is a method for preparing a beverage comprising (I) providing a beverage base and (ii) adding rebaudioside I to the consumable base to provide a beverage.

In another aspect, the present invention is a method of preparing a sweetened consumable comprising (I) providing a sweetenable consumable and (ii) adding rebaudioside I to the sweetenable consumable to provide a sweetened consumable.

In a particular embodiment, the present invention is a method of preparing a sweetened beverage comprising (I) providing a sweetenable beverage and (ii) adding rebaudioside I to the sweetenable beverage to provide a sweetened beverage.

In the above methods, rebaudioside I can be provided as such, or in the form of a composition. When rebaudioside I is provided as a composition, the amount of the composition is effective to provide a rebaudioside I concentration that is above, at, or below its threshold flavor or sweetness recognition concentration when the composition is added to a consumable (e.g., a beverage). When rebaudioside I is not provided as a composition, it can be added to the consumable at a concentration that is above, at, or below its threshold flavor or sweetness recognition concentration.

In one embodiment, the present invention is a method for enhancing the sweetness of a consumable comprising (I) providing a consumable comprising one or more sweet ingredients, and (ii) adding rebaudioside I (1) to the consumable to provide a consumable with enhanced sweetness, wherein rebaudioside I is added to the consumable at a concentration at or below its threshold sweetness recognition concentration. In a particular embodiment, rebaudioside I is added to the consumable at a concentration below its threshold sweetness recognition concentration.

In another embodiment, the present invention is a method of enhancing the sweetness of a consumable comprising (I) providing a consumable comprising one or more sweet tasting ingredients, and (ii) adding a composition comprising rebaudioside I to the consumable to provide a consumable with enhanced sweetness, wherein rebaudioside I is present in the composition in an amount effective to provide a concentration of rebaudioside I at or below its threshold sweetness recognition concentration when the composition is added to the consumable. In a particular embodiment, rebaudioside I is present in the composition in an amount effective to provide a rebaudioside I concentration below its threshold sweetness recognition concentration.

In a particular embodiment, the present invention is a method for enhancing sweetness of a beverage comprising (I) providing a beverage comprising at least one sweet taste ingredient, and (ii) adding rebaudioside I to the beverage to provide a beverage having enhanced sweetness, wherein rebaudioside I is added to the beverage in an amount effective to provide a concentration at or below its threshold sweetness recognition concentration. In a particular embodiment, rebaudioside I is added to the consumable in an amount effective to provide a concentration below its threshold sweetness recognition concentration.

In another particular embodiment, the present invention is a method of enhancing the sweetness of a beverage comprising (I) providing a beverage comprising one or more sweet tasting ingredients, and (ii) adding a composition comprising rebaudioside I to the beverage to provide a beverage having enhanced sweetness, wherein rebaudioside I is present in the composition in an amount effective to provide a concentration of rebaudioside I at or below its threshold sweetness recognition concentration when the composition is added to the beverage. In particular embodiments, rebaudioside I is present in the composition in an amount effective to provide a rebaudioside I concentration below its threshold sweetness recognition concentration when the composition is added to a beverage.

In another embodiment, the present invention is a method for enhancing the flavor of a consumable comprising (I) providing a consumable comprising at least one flavor ingredient, and (ii) adding rebaudioside I to the consumable to provide a consumable with enhanced flavor, wherein rebaudioside I is added to the consumable at a concentration at or below its threshold flavor recognition concentration. In a particular embodiment, rebaudioside I is added to the consumable at a concentration below the threshold flavor recognition concentration.

In another embodiment, the present invention is a method for enhancing the flavor of a consumable comprising (I) providing a consumable comprising at least one flavor ingredient, and (ii) adding rebaudioside I to the consumable to provide a consumable with enhanced flavor, wherein rebaudioside I is present in the composition in an amount effective to provide a concentration of rebaudioside I at or below its threshold flavor recognition concentration when the composition is added to the consumable. In a particular embodiment, rebaudioside I is present in the composition in an amount effective to provide a rebaudioside I concentration below its threshold flavor recognition concentration when the composition is added to the consumable.

In a particular embodiment, the present invention is a method for enhancing the flavor of a beverage comprising (I) providing a beverage comprising at least one flavor ingredient, and (ii) adding rebaudioside I to the beverage to provide a beverage having an enhanced flavor, wherein rebaudioside I is added to the beverage at a concentration at or below its threshold flavor recognition concentration. In a particular embodiment, rebaudioside I is added to the consumable at a concentration below the threshold flavor recognition concentration.

In a particular embodiment, the present invention is a method for enhancing the flavor of a beverage comprising (I) providing a beverage comprising at least one flavor ingredient, and (ii) adding a composition comprising rebaudioside I to the beverage to provide a beverage having an enhanced flavor, wherein rebaudioside I is present in the composition in an amount effective to provide a concentration of rebaudioside I at or below its threshold flavor recognition concentration when the composition is added to the beverage. In a particular embodiment, rebaudioside I is present in the composition in an amount effective to provide a rebaudioside I concentration below its threshold flavor recognition concentration when the composition is added to the consumable.

The present invention also includes methods of preparing sweetened compositions (e.g., sweetened consumables) and flavor enhancing compositions (e.g., flavor enhancing consumables) by adding rebaudioside I or compositions comprising rebaudioside I to such compositions/consumables.

The following examples illustrate preferred embodiments of the invention. It is to be understood that this invention is not limited to the materials, proportions, conditions, and procedures set forth in these examples, which are illustrative only.

Example 1

In vivo production of UGT76G1

Restriction sites NcoI and NdeI were added to the original nucleic acid sequence described in Genbank accession No. AAR 06912.1. After codon optimization, the following nucleic acid sequences were obtained:

CCATGGCCCATATGGAAAACAAAACCGAAACCACCGTTCGTCGTCGTCGCCGT ATTATTCTGTTTCCGGTTCCGTTTCAGGGTCATATTAATCCGATTCTGCAGCTG GCAAATGTGCTGTATAGCAAAGGTTTTAGCATTACCATTTTTCATACCAATTTT AACAAACCGAAAACCAGCAATTATCCGCATTTTACCTTTCGCTTTATTCTGGAT AATGATCCGCAGGATGAACGCATTAGCAATCTGCCGACACATGGTCCGCTGGC AGGTATGCGTATTCCGATTATTAACGAACATGGTGCAGATGAACTGCGTCGTG AACTGGAACTGCTGATGCTGGCAAGCGAAGAAGATGAAGAAGTTAGCTGTCT GATTACCGATGCACTGTGGTATTTTGCACAGAGCGTTGCAGATAGCCTGAATC TGCGTCGTCTGGTTCTGATGACCAGCAGCCTGTTTAACTTTCATGCACATGTTA GCCTGCCGCAGTTTGATGAACTGGGTTATCTGGATCCGGATGATAAAACCCGT CTGGAAGAACAGGCAAGCGGTTTTCCGATGCTGAAAGTGAAAGATATCAAAA GCGCCTATAGCAATTGGCAGATTCTGAAAGAAATTCTGGGCAAAATGATTAAA CAGACCAAAGCAAGCAGCGGTGTTATTTGGAATAGCTTTAAAGAACTGGAAG AAAGCGAACTGGAAACCGTGATTCGTGAAATTCCGGCACCGAGCTTTCTGATT CCGCTGCCGAAACATCTGACCGCAAGCAGCAGCAGCCTGCTGGATCATGATCG TACCGTTTTTCAGTGGCTGGATCAGCAGCCTCCGAGCAGCGTTCTGTATGTTAG CTTTGGTAGCACCAGCGAAGTTGATGAAAAAGATTTTCTGGAAATTGCCCGTG GTCTGGTTGATAGCAAACAGAGCTTTCTGTGGGTTGTTCGTCCGGGTTTTGTTA AAGGTAGCACCTGGGTTGAACCGCTGCCGGATGGTTTTCTGGGTGAACGTGGT CGTATTGTTAAATGGGTTCCGCAGCAAGAAGTTCTGGCACACGGCGCAATTGG TGCATTTTGGACCCATAGCGGTTGGAATAGCACCCTGGAAAGCGTTTGTGAAG GTGTTCCGATGATTTTTAGCGATTTTGGTCTGGATCAGCCGCTGAATGCACGTT ATATGAGTGATGTTCTGAAAGTGGGTGTGTATCTGGAAAATGGTTGGGAACGT GGTGAAATTGCAAATGCAATTCGTCGTGTTATGGTGGATGAAGAAGGTGAATA TATTCGTCAGAATGCCCGTGTTCTGAAACAGAAAGCAGATGTTAGCCTGATGA AAGGTGGTAGCAGCTATGAAAGCCTGGAAAGTCTGGTTAGCTATATTAGCAGC CTGTAATAACTCGA(SEQ ID NO：1)。

after synthesizing the gene and subcloning the gene into the pET30A + vector using NdeI and XhoI cloning sites, the UGT76G1_ pET30a + plasmid was introduced into escherichia coli Bl21(DE3) and escherichia coli EC100 by electroporation. The obtained cells were cultured in a petri dish in the presence of kanamycin, and appropriate colonies were selected and allowed to grow in liquid LB medium (erlenmeyer flask). Glycerol was added to the suspension as a cryoprotectant and 400 μ Ι _ aliquots were stored at-20 ℃ and at-80 ℃.

A stock aliquot of E.coli BL21(DE3) containing the pET30A + _ UGT76G1 plasmid was thawed and added to 30mL of LBGKP medium (20G/L Luria Broth Lennox; 50mM PIPES buffer pH 7.00; 50mM phosphate buffer pH 7.00; 2.5G/L glucose and 50mg/L kanamycin). This incubation was carried out at 30 ℃ for 8h with shaking at 135 rpm.

The production medium contained 60g/L of overnight fast expression TB medium (Novagen), 10g/L of glycerol and 50mg/L of kanamycin. The medium was stirred at 20 ℃ while samples were taken for OD and pH measurements. The culture produced significant growth and a good OD was obtained. After 40 hours, cells were harvested by centrifugation and frozen to yield a wet weight of 12.7g cells.

Lysis was performed by adding a Bugbuster master mix (Novagen Co.), and the lysate was recovered by centrifugation and kept frozen. The thawed lysates were tested for activity.

Example 2

In vitro production of UGT76G1

A S30T7 high yield protein expression system kit from Promega (Promega) was used. Mu.g of UGT76G1_ pET30a + plasmid from E.coli EC100 was mixed with 80. mu.L of S30 premix plus, and 72. mu.L of S30T7 extract was added. Nuclease-free water was added to obtain a total volume of 200. mu.L, and the resulting solution was incubated at 30 ℃ for 2 h. 180 μ L was used for the catalytic test reaction.

Example 3

In vitro production of UGT91D2

Restriction sites NcoI and NdeI were added to the original nucleic acid sequence described in Genbank accession number ACE 87855.1. After codon optimization, the following nucleic acid sequences were obtained:

CCATGGCACATATGGCAACCAGCGATAGCATTGTTGATGATCGTAAACAGCTG CATGTTGCAACCTTTCCGTGGCTGGCATTTGGTCATATTCTGCCGTATCTGCAG CTGAGCAAACTGATTGCAGAAAAAGGTCATAAAGTGAGCTTTCTGAGCACCA CCCGTAATATTCAGCGTCTGAGCAGCCATATTAGTCCGCTGATTAATGTTGTTC AGCTGACCCTGCCTCGTGTTCAAGAACTGCCGGAAGATGCCGAAGCAACCACC GATGTTCATCCGGAAGATATTCCGTATCTGAAAAAAGCAAGTGATGGTCTGCA GCCGGAAGTTACCCGTTTTCTGGAACAGCATAGTCCGGATTGGATCATCTATG ATTATACCCATTATTGGCTGCCGAGCATTGCAGCAAGCCTGGGTATTAGCCGT GCACATTTTAGCGTTACCACCCCGTGGGCAATTGCATATATGGGTCCGAGCGC AGATGCAATGATTAATGGTAGTGATGGTCGTACCACCGTTGAAGATCTGACCA CCCCTCCGAAATGGTTTCCGTTTCCGACCAAAGTTTGTTGGCGTAAACATGATC TGGCACGTCTGGTTCCGTATAAAGCACCGGGTATTAGTGATGGTTATCGTATG GGTCTGGTTCTGAAAGGTAGCGATTGTCTGCTGAGCAAATGCTATCATGAATT TGGCACCCAGTGGCTGCCGCTGCTGGAAACCCTGCATCAGGTTCCGGTTGTTC CGGTGGGTCTGCTGCCTCCGGAAGTTCCGGGTGATGAAAAAGATGAAACCTG GGTTAGCATCAAAAAATGGCTGGATGGTAAACAGAAAGGTAGCGTGGTTTAT GTTGCACTGGGTAGCGAAGTTCTGGTTAGCCAGACCGAAGTTGTTGAACTGGC ACTGGGTCTGGAACTGAGCGGTCTGCCGTTTGTTTGGGCATATCGTAAACCGA AAGGTCCGGCAAAAAGCGATAGCGTTGAACTGCCGGATGGTTTTGTTGAACGT ACCCGTGATCGTGGTCTGGTTTGGACCAGCTGGGCACCTCAGCTGCGTATTCT GAGCCATGAAAGCGTTTGTGGTTTTCTGACCCATTGTGGTAGCGGTAGCATTG TGGAAGGTCTGATGTTTGGTCATCCGCTGATTATGCTGCCGATTTTTGGTGATC AGCCGCTGAATGCACGTCTGCTGGAAGATAAACAGGTTGGTATTGAAATTCCG CGTAATGAAGAAGATGGTTGCCTGACCAAAGAAAGCGTTGCACGTAGCCTGC GTAGCGTTGTTGTTGAAAAAGAAGGCGAAATCTATAAAGCCAATGCACGTGA ACTGAGCAAAATCTATAATGATACCAAAGTGGAAAAAGAATATGTGAGCCAG TTCGTGGATTATCTGGAAAAAAACACCCGTGCAGTTGCCATTGATCACGAAAG CTAATGACTCGAG(SEQ ID NO：2)。

after synthesizing the gene and subcloning it into the pET30A + vector using the NcoI and XhoI cloning sites, the UGT91D2_ pET30a + plasmid was introduced into e.coli EC100 by electroporation. The cells obtained were cultured in the presence of kanamycin, and appropriate colonies were selected and allowed to grow in liquid LB medium (erlenmeyer flask). Glycerol was added to the suspension as a cryoprotectant and 400 μ Ι _ aliquots were stored at-20 ℃ and at-80 ℃.

The S30T7 high yield protein expression system kit from promegate was used for in vitro protein synthesis.

Mu.g of UGT91D2_ pET30a + plasmid was mixed with 80. mu.L of S30 premix plus, and 72. mu.L of S30T7 extract was added. Nuclease-free water was added to obtain a total volume of 200. mu.L, and the resulting solution was incubated at 30 ℃ for 2 h. mu.L was used for SDS-page analysis, while the remaining 45. mu.L was used in the catalytic test reaction.

Example 4

In vivo production of UGTSL

The pET30A + vector containing the gene corresponding to the enzyme (GI 460409128, Version XP-004249992.1) was introduced into E.coli BL21(DE3) by heat shock. The obtained cells were cultured in a petri dish in the presence of kanamycin, and appropriate colonies were selected and allowed to grow in liquid LB medium (erlenmeyer flask). Glycerol was added to the suspension as a cryoprotectant and 400 μ Ι _ aliquots were stored at-20 ℃ and at-80 ℃.

A stock aliquot of E.coli BL21(DE3) containing pET30A + _ UGT plasmid was thawed and added to 30mL of LBGKP medium (20g/L Luria Broth Lennox; 50mM PIPES buffer pH 7.00; 50mM phosphate buffer pH 7.00; 2.5g/L glucose and 50mg/L kanamycin). This incubation was carried out at 30 ℃ for 8 hours under shaking at 135 rpm.

The production medium contained 60g/L of overnight fast expression TB medium (Novagen), 10g/L of glycerol and 50mg/L of kanamycin. The pre-culture was added to 400mL of the medium and the solution was stirred at 20 ℃ while a sample was taken to measure OD and pH. The culture produced significant growth and a good OD was obtained. After 40 hours, cells were harvested by centrifugation and frozen. The wet weight of the Cells (CWW) was 6.8 g.

Lysis was performed by adding a Bugbuster master mix (Novagen Co.), and the lysate was recovered by centrifugation and used fresh.

Example 5

In vivo production of UGTSL2

UGTSL2(GI _460410132/XP _004250485.1) amino acid sequence:

MATNLRVLMFPWLAYGHISPFLNIAKQLADRGFLIYLCSTRINLESIIKKIPEKYAD SIHLIELQLPELPELPPHYHTTNGLPPHLNPTLHKALKMSKPNFSRILQNLKPDLLIY DVLQPWAEHVANEQNIPAGKLLTSCAAVFSYFFSFRKNPGVEFPFPAIHLPEVEKV KIREILAKEPEEGGRLDEGNKQMMLMCTSRTIEAKYIDYCTELCNWKVVPVGPPF QDLITNDADNKELIDWLGTKHENSTVFVSFGSEYFLSKEDMEEVAFALELSNVNFI WVARFPKGEERNLEDALPKGFLERIGERGRVLDKFAPQPRILNHPSTGGFISHCGW NSAMESIDFGVPIIAMPIHNDQPINAKLMVELGVAVEIVRDDDGKIHRGEIAETLKS VVTGETGEILRAKVREISKNLKSIRDEEMDAVAEELIQLCRNSNKSK(SEQ ID NO： 9)。

the pET30A + vector containing the UGTSL2 gene was introduced into E.coli B121 (DE3) by heat shock. The obtained cells were cultured in a petri dish in the presence of kanamycin, and appropriate colonies were selected and allowed to grow in liquid LB medium (erlenmeyer flask). Glycerol was added to the suspension as a cryoprotectant and 400 μ Ι _ aliquots were stored at-20 ℃ and at-80 ℃.

A stock aliquot of E.coli BL21(DE3) containing the pET30A + _ UGTSL2 plasmid was thawed and added to 30mL of LBGKP medium (20g/L Luria Broth Lennox; 50mM PIPES buffer pH 7.00; 50mM phosphate buffer pH 7.00; 2.5g/L glucose and 50mg/L kanamycin). This incubation was carried out at 30 ℃ for 8h with shaking at 135 rpm.

The production medium contained 60g/L of overnight fast expression TB medium (Novagen), 10g/L of glycerol and 50mg/L of kanamycin. Preculture was added to 200mL of the medium and the solution was stirred at 20 ℃ while samples were taken to measure OD and pH. The culture produced significant growth and a good OD was obtained. After 40 hours, the cells were harvested by centrifugation, frozen and 6.22g of wet weight of the cells was obtained.

Lysis was performed by adding Bugbuster master mix (Novagen) to 1.4g of cells, and the lysate was recovered by centrifugation and used fresh.

Example 6

Catalytic reactions with in vivo generated UGT76G1

The total reaction volume was 5.0mL, with the following composition: 50mM sodium phosphate buffer pH 7.2, 3mM MgCl₂2.5mM UDP-glucose, 0.5mM stevioside and 500. mu.L of UGT76G 1. The reaction was run on an orbital shaker at 135rpm at 30 ℃. For each sample, 40. mu.L of 2N H was used ₂SO₄And 420. mu.L of methanol/water (6/4) 460. mu.L of the reaction mixture was quenched. The samples were immediately centrifuged and maintained at 10 ℃ before analysis by hplc (cad). HPLC indicated almost complete conversion of stevioside to rebaudioside a (fig. 4).

Example 7

Preparation and Activity of UGT76G1 prepared by pET30a + plasmid and BL21(DE3) expression Strain

pET30a + _ UGT76G1 plasmid was transformed into BL21DE3) expression strain (Lucigen E.

EXPRESS electrocompetent cells). The obtained cells were grown on LB agar medium in a petri dish in the presence of kanamycin. SelectingSuitable colonies were grown in liquid LBGKP medium containing kanamycin. Glycerol was added and 400 μ L aliquots were stored at-20 ℃ and at-80 ℃.

Storage aliquots were thawed and added to 30mL of LBGKP medium. This culture was carried out for 8 hours at 30 ℃ with shaking, and then it was used to inoculate 400mL of a production medium containing 60g/L of "overnight Rapid expression TB medium" (Novagen, ref 71491-5), 10g/L of glycerol and 50mg/L of kanamycin. The medium was stirred at 20 ℃ while samples were taken for OD (600nm) and pH measurements. After 40 hours, cells were harvested by centrifugation and frozen. The wet weight of the cells obtained was 10.58 g.

3.24g of the obtained precipitate was lysed by adding 8.1mL of "Bugbuster master mix" (Novagen Co., reference 71456) and 3.5mL of water. The lysate was recovered by centrifugation and kept frozen.

Example 8

Preparation and Activity of UGT76G1 prepared by pET30a + plasmid and Tuner (DE3) expression Strain

The pET30a + _ UGT76G1 plasmid was transformed into a Tuner (DE3) expression strain (Novagen Tuner) by heat shock treatment^tm(DE3) competent cells). The obtained cells were grown on LB agar medium in a petri dish in the presence of kanamycin. Appropriate colonies were selected and grown in liquid LBGKP medium containing kanamycin). Glycerol was added and 400 μ L aliquots were stored at-20 ℃ and at-80 ℃.

Storage aliquots were thawed and added to 100mL LB medium containing 50mg/L kanamycin. This culture was carried out at 30 ℃ for 15h with shaking. 4.4mL of this culture was used to inoculate 200mL of production medium containing LB. The medium was stirred at 37 ℃ until an OD of 0.9 (600nm) was obtained, followed by addition of 400. mu.L of a 100mM IPTG solution and stirring of the medium at 30 ℃ for 4 hours. Cells were harvested by centrifugation and frozen. The wet weight of the cells obtained was 1.38 g.

The obtained precipitate was lysed by adding 4.9mL of "Bugbuster master mix" (Novagen Co., reference 71456) and 2.1mL of water. The lysate was recovered by centrifugation and kept frozen.

Example 9

Preparation and Activity of UGT76G1 prepared by pMAL plasmid and BL21 expression Strain

After subcloning the synthetic UGT76G1 gene into the pMAL plasmid using Nde1 and Sal1 cloning sites, the pMAL _ UGT76G1 plasmid was transformed into the BL21 expression strain (New England Biolabs BL21 competent E.coli) by heat shock treatment. The obtained cells were grown on LB agar medium in a petri dish in the presence of ampicillin. Suitable colonies were selected and grown in liquid LBGKP medium containing ampicillin). Glycerol was added and 400 μ L aliquots were stored at-20 ℃ and at-80 ℃.

Storage aliquots were thawed and added to 30mL of LBGKP medium. This culture was carried out for 8h at 30 ℃ with shaking, and then it was used to inoculate 400mL of a production medium containing 60g/L of "overnight Rapid expression TB medium" (Novagen, ref 71491-5), 10g/L of glycerol and 50mg/L of ampicillin. The medium was stirred at 20 ℃ while samples were taken for OD and pH measurements. After 40 hours, cells were harvested by centrifugation and frozen. The wet weight of the cells obtained was 5.86 g.

2.74g of the obtained precipitate was lysed by adding 9.6mL of "Bugbuster master mix" (Novagen Co., reference 71456) and 4.1mL of water. The lysate was recovered by centrifugation and kept frozen.

Example 10

Preparation and Activity of UGT76G1 prepared by pMAL plasmid and ArcticExpress expression Strain

The pMAL _ UGT76G1 plasmid was transformed into an Articexpress expression strain (Agilent ArcticExpress competent cells) by heat shock treatment. The obtained cells were grown on LB agar medium in a petri dish in the presence of ampicillin and geneticin. Suitable colonies were selected and grown in liquid LBGKP medium containing ampicillin and geneticin. Glycerol was added and 400 μ L aliquots were stored at-20 ℃ and at-80 ℃.

Storage aliquots were thawed and added to 30mL of LBGKP medium (containing ampicillin and geneticin). This culture was carried out for 8h at 30 ℃ with shaking, and then it was used to inoculate 400mL of a production medium containing 60g/L of "overnight Rapid expression TB medium" (Novagen, ref 71491-5), 10g/L of glycerol and 50mg/L of ampicillin. The medium was stirred at 12 ℃ while samples were taken for OD (600nm) and pH measurements. After 68 hours, cells were harvested by centrifugation and frozen. The wet weight of the cells obtained was 8.96 g.

2.47g of the obtained precipitate was lysed by adding 8.73mL of "Bugbuster master mix" (Novagen Co., reference 71456) and 3.79mL of water. The lysate was recovered by centrifugation and kept frozen.

Example 11

Preparation and Activity of UGT76G1 prepared by pCOLDIII plasmid and ArcticExpress expression Strain

After subcloning the synthetic UGT76G1 gene into the pCOLDIII plasmid using Nde1 and Xho1 cloning sites, the pCOLDIII _ UGT76G1 plasmid was transformed into an ArcticExpress expressing strain (agilent ArcticExpress competent cells) by heat shock treatment. The obtained cells were grown on LB agar medium in a petri dish in the presence of ampicillin and geneticin. Suitable colonies were selected and grown in liquid LBGKP medium containing ampicillin and geneticin. Glycerol was added and 400 μ L aliquots were stored at-20 ℃ and at-80 ℃.

Storage aliquots were thawed and added to 30mL of LBGKP medium (containing ampicillin and geneticin). This culture was carried out for 8 hours at 30 ℃ with shaking, and then it was used to inoculate 400mL of a production medium containing 60g/L of "overnight Rapid expression TB medium" (Novagen, ref 71491-5), 10g/L of glycerol and 50mg/L of kanamycin. The medium was stirred at 12 ℃ while samples were taken for OD (600nm) and pH measurements. After 63 hours, the cells were harvested by centrifugation and frozen. The wet weight of the cells obtained was 6.54 g.

2.81g of the obtained precipitate was lysed by adding 9.8mL of "Bugbuster master mix" (Novagen Co., reference 71456) and 4.2mL of water. The lysate was recovered by centrifugation and kept frozen.

Example 12

Preparation and Activity of UGT76G1 prepared by pCOLDIII plasmid and Origami2(DE3) expression Strain

The pCOLDIII _ UGT76G1 plasmid was transformed into Origami2(DE3) expressing strain (Novagen Origami) by heat shock treatment^TM2(DE3) competent cells). The obtained cells were grown on LB agar medium in a petri dish in the presence of ampicillin. Suitable colonies were selected and grown in liquid LBGKP medium containing ampicillin. Glycerol was added and 400 μ L aliquots were stored at-20 ℃ and at-80 ℃.

Storage aliquots were thawed and added to 30mL of LBGKP medium (containing ampicillin). This culture was carried out for 8 hours at 30 ℃ with shaking, and then it was used to inoculate 400mL of a production medium containing 60g/L of "overnight Rapid expression TB medium" (Novagen, ref 71491-5), 10g/L of glycerol and 50mg/L of kanamycin. The medium was stirred at 12 ℃ while samples were taken for OD (600nm) and pH measurements. After 68 hours, cells were harvested by centrifugation and frozen. The wet weight of the cells obtained was 2.53 g.

1.71g of the obtained precipitate was lysed by adding 6.0mL of "Bugbuster master mix" (Novagen Co., reference 71456) and 1.9mL of water. The lysate was recovered by centrifugation and kept frozen.

Example 13

UGT91D2 was prepared using pMAL plasmid and BL21 expression strain

After subcloning the synthetic UGT91D2 gene into the pMAL plasmid using Nde1 and Sal1 cloning sites, the pMAL _ UGT91D2 plasmid was transformed into the BL21 expression strain (New England Biolabs BL21 competent E.coli) by heat shock treatment. The obtained cells were grown on LB agar medium in a petri dish in the presence of ampicillin. Suitable colonies were selected and grown in liquid LBGKP medium containing ampicillin). Glycerol was added and 400 μ L aliquots were stored at-20 ℃ and at-80 ℃.

Storage aliquots were thawed and added to 30mL of LBGKP medium. This culture was carried out for 8h at 30 ℃ with shaking, and then it was used to inoculate 400mL of a production medium containing 60g/L of "overnight Rapid expression TB medium" (Novagen, ref 71491-5), 10g/L of glycerol and 50mg/L of ampicillin. The medium was stirred at 20 ℃ while samples were taken for OD and pH measurements. After 40 hours, cells were harvested by centrifugation and frozen. The wet weight of the cells obtained was 12.32 g.

2.18g of the obtained precipitate were lysed by adding 7.7mL of "Bugbuster master mix" (Novagen Co., reference 71456) and 3.2mL of water. The lysate was recovered by centrifugation and used directly for activity testing.

Example 14

Preparation of UGT91D2 Using pMAL plasmid and ArcticExpress expression Strain

The pMAL _ UGT91D2 plasmid was transformed into an ArcticExpress expression strain (agilent ArcticExpress competent cells) by heat shock treatment. The obtained cells were grown on LB agar medium in a petri dish in the presence of ampicillin and geneticin. Suitable colonies were selected and grown in liquid LBGKP medium containing ampicillin and geneticin. Glycerol was added and 400 μ L aliquots were stored at-20 ℃ and at-80 ℃.

Storage aliquots were thawed and added to 30mL of LBGKP medium (containing ampicillin and geneticin). This culture was carried out for 8h at 30 ℃ with shaking, and then it was used to inoculate 400mL of a production medium containing 60g/L of "overnight Rapid expression TB medium" (Novagen, ref 71491-5), 10g/L of glycerol and 50mg/L of ampicillin. The medium was stirred at 20 ℃ for 16 hours, then at 12 ℃ for an additional 50 hours, while samples were taken to measure OD (600nm) and pH. Cells were harvested by centrifugation and frozen. The wet weight of the cells obtained was 15.77 g.

2.57g of the obtained precipitate was lysed by adding 9.0mL of "Bugbuster master mix" (Novagen Co., reference 71456) and 3.8mL of water. The lysate was recovered by centrifugation and used directly for activity testing.

Example 15

UGT91D2 was prepared using pET30a + plasmid and Tuner (DE3) expression strain

The pET30a + _ UGT91D2 plasmid was transformed into a Tuner (DE3) expression strain (Novagen Tuner) by heat shock treatment^tm(DE3) competent cells). The obtained cells were grown on LB agar medium in a petri dish in the presence of kanamycin. Suitable colonies were selected and grown in liquid LBGKP medium (containing kanamycin). Glycerol was added and 400 μ L aliquots were stored at-20 ℃ and at-80 ℃.

Storage aliquots were thawed and added to 100mL LB medium containing 50mg/L kanamycin. This culture was carried out at 30 ℃ for 15h with shaking. 6.2mL of this culture was used to inoculate 500mL of production medium containing LB. The medium was stirred at 37 ℃ until an OD (600nm) of 0.9 was obtained, followed by addition of 500. mu.L of a 100mM IPTG solution (IPTG concentration in the medium of 100. mu.M), and stirring of the medium at 30 ℃ for 4 hours, and the cells were harvested by centrifugation and frozen. The wet weight of the cells obtained was 4.02 g.

1.92g of the obtained precipitate was lysed by adding 6.8mL of "Bugbuster master mix" (Novagen Co., reference 71456) and 2.8mL of water. Lysates were recovered by centrifugation and directly subjected to activity assays.

Example 16

Preparation of UGT91D2 Using pET30a + plasmid and ArcticExpress expression Strain

The pET30a + _ UGT91D2 plasmid was transformed into an ArcticExpress (DE3) expressing strain (Agilent ArcticExpress competent cells) by heat shock treatment. The obtained cells were grown on LB agar medium in a petri dish in the presence of kanamycin and geneticin. Suitable colonies were selected and grown in liquid LBGKP medium containing kanamycin and geneticin. Glycerol was added and 400 μ L aliquots were stored at-20 ℃ and at-80 ℃.

Storage aliquots were thawed and added to 30mL of LBGKP medium (containing kanamycin and geneticin). This culture was carried out for 8h at 30 ℃ with shaking, and then it was used to inoculate 400mL of a production medium containing 60g/L of "overnight Rapid expression TB medium" (Novagen, ref 71491-5), 10g/L of glycerol and 50mg/L of ampicillin. The medium was stirred at 20 ℃ for 16 hours. Then at 12 ℃ for another 50 hours. Samples were taken at the same time to measure OD (600nm) and pH. After 60 hours, cells were harvested by centrifugation and frozen. The wet weight of the cells obtained was 16.07 g.

3.24g of the obtained precipitate was lysed by adding 11.4mL of "Bugbuster master mix" (Novagen Co., reference 71456) and 4.8mL of water. The lysate was recovered by centrifugation and used directly for activity testing.

Example 17

Activity assay for in vivo formulations of UGT91D2

Using 0.5mM substrate, 2.5mM UDP-glucose and 3mM MgCl in 50mM sodium phosphate buffer pH 7.2₂Activity test on conversion of rubusoside to stevioside was performed on a 5mL scale using 1000 μ L of lysate. Samples were taken and analyzed by HPLC. The results of the different preparations of UGT91D2 are summarized in the table below.

Note that: activity is mentioned per mL of lysate. 1U is the substrate which has been converted to 1. mu. mol in 1 hour at 30 ℃ and pH 7.2.

Example 18

Directed evolution of UGT76G1 for conversion of rebaudioside D to rebaudioside M

Starting from the amino acid sequence of UGT76G1 as described in Genbank (AAR06912.1), different mutations at various amino acid positions were identified that could alter the activity of the enzyme for the conversion of rebaudioside d (reb d) to rebaudioside m (reb m). By DNA2.0ProteinGPS^TMThis list of strategically designed mutations was then used to synthesize 96 variant genes containing 3, 4 or 5 of these mutations that were codon optimized for expression in e. These genes were subcloned into the pET30a + plasmid and used to transform E.coli BL21(DE3) chemically competent cells. The cells obtained were subjected to kanamycin The plates were grown on solid LB agar medium. Suitable colonies were selected and grown in liquid LB medium in test tubes. Glycerol was added to the suspension as a cryoprotectant and 400 μ Ι _ aliquots were stored at-20 ℃ and at-80 ℃.

These storage aliquots of E.coli BL21(DE3) containing the pET30a + _ UGT76G1var plasmid were thawed and added to LBGKP medium (20G/L Luria Broth Lennox; 50mM PIPES buffer pH 7.00; 50mM phosphate buffer pH 7.00; 2.5G/L glucose and 50mg/L kanamycin). This culture was carried out in 96 microtiter plates at 135rpm at 30 ℃ for 8h with shaking.

50 μ L of the above culture was inoculated into Overnight fast expression TB medium (Overnight Express) containing 60g/L^TM Instant TB medium)

10g/L glycerol and 50mg/L kanamycin in 3.95mL of production medium. The resulting culture was stirred at 20 ℃ in a 48-deep well plate. The culture produced significant growth and a good OD (600 nm; 1cm) was obtained. After 44 hours, cells were harvested by centrifugation and frozen.

By adding to thawed cells

Masterbatch

Lysis was performed and the lysate was recovered by centrifugation. Activity testing was performed with 100. mu.L of fresh lysate added to rebaudioside D (final concentration 0.5mM), MgCl, in 50mM phosphate buffer (pH 7.2) ₂(final concentration 3mM) and UDP-glucose (final concentration 2.5 mM).

The reaction was allowed to proceed at 30 ℃ and samples were taken after 2, 4, 7 and 24 hours to determine conversion and initial rate by HPLC (CAD detection) using the analytical methods described above for conversion of rebaudioside D to rebaudioside M. The results are depicted in the following table.

Mutations are labeled as follows: original amino acid-position-new amino acid: for example, the mutation of alanine to glycine at position 33 is labeled a 33G.

Example 19

In vivo production of UGT76G1 in Saccharomyces cerevisiae

UGT76G1[ stevia rebaudiana (gi _37993653/gb _ AAR06912.1)

MENKTETTVRRRRRIILFPVPFQGHINPILQLANVLYSKGFSITIFHTNFNKPKTSNY PHFTFRFILDNDPQDERISNLPTHGPLAGMRIPIINEHGADELRRELELLMLASEEDE EVSCLITDALWYFAQSVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDD KTRLEEQASGFPMLKVKDIKSAYSNWQILKEILGKMIKQTKASSGVIWNSFKELEE SELETVIREIPAPSFLIPLPKHLTASSSSLLDHDRTVFQWLDQQPPSSVLYVSFGSTS EVDEKDFLEIARGLVDSKQSFLWVVRPGFVKGSTWVEPLPDGFLGERGRIVKWVP QQEVLAHGAIGAFWTHSGWNSTLESVCEGVPMIFSDFGLDQPLNARYMSDVLKV GVYLENGWERGEIANAIRRVMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESL ESLVSYISSL。

The above amino acid sequence was codon optimized for expression in Saccharomyces cerevisiae. In addition, the yeast consensus sequence AACACA was added before the ATG start codon. The synthetic gene was subcloned into the pYES2 vector using Hind III and Xba I restriction sites. The pYES2_ UGT76G1_ Sc vector was used to transform chemically competent Saccharomyces cerevisiae INVSC1 cells (Invitrogen).

Cells were grown on uracil-deficient solid synthetic minimal medium containing 2% glucose, and individual colonies were picked and grown on uracil-deficient liquid synthetic minimal medium (SC-U containing 2% glucose). After centrifugation, cells were suspended with SC-U (containing 2% glucose) and 60% glycerol/water. Aliquots were stored at-80 ℃ and one aliquot was used to start incubation in SC-U (containing 2% glucose) for 43 hours at 30 ℃. A portion of this culture was centrifuged and suspended in induction medium (SC-U containing 2% galactose) for 19 hours at 30 ℃.

Cells were obtained by centrifugation and five volumes of CelLytic were used^TMY cell lysis reagent (sigma) was used for lysis. The lysate was used directly for activity testing (UGT76G1_ Sc).

Example 20

Directed evolution of UGT76G1 for conversion of rebaudioside D to rebaudioside M (round 2)

The most active clone from the first round directed evolution of UGT76G1 (see example 18, UGT76G1var94 containing the mutation Q266E _ P272A _ R334K _ G348P _ L379G) was selected as the baseline clone for round 2. A list of 53 mutations was created containing the different identified positive mutations from the first round and the new mutations obtained by the dna2.0protein gpstm strategy. This list of mutations was then used to design 92 variant genes, each containing 3 different mutations. After codon optimization for expression in e.coli, these genes were synthesized, subcloned into the pET30a + plasmid and used to transform e.coli BL21(DE3) chemically competent cells. The obtained cells were grown in a petri dish on solid LB agar medium in the presence of kanamycin. Suitable colonies were selected and grown in liquid LB medium in test tubes. Glycerol was added to the suspension as a cryoprotectant and 400 μ Ι _ aliquots were stored at-20 ℃ and at-80 ℃.

These storage aliquots of E.coli BL21(DE3) containing the pET30a + _ UGT76G1var plasmid were thawed and added to LBGKP medium (20G/L Luria Broth Lennox; 50mM PIPES buffer pH 7.00; 50mM phosphate buffer pH 7.00; 2.5G/L glucose and 50mg/L kanamycin). This incubation was performed in microtiter plates at 30 ℃ for 8h with shaking.

50 μ L of the above culture was inoculated into Overnight fast expression TB medium (Overnight Express) containing 60g/L^TM Instant TB medium)

10g/L glycerol and 50mg/L kanamycin in 3.95mL of production medium. The resulting culture was stirred at 20 ℃ in a 48-deep well plate. The culture produced significant growth and a good OD (600nm) was obtained. After 44 hours, cells were harvested by centrifugation and frozen.

By adding to thawed cells

Masterbatch

Lysis was performed and the lysate was recovered by centrifugation. Activity testing was performed with 100. mu.L of fresh lysate added to rebaudioside D (final concentration 0.5mM), MgCl, in 50mM phosphate buffer (pH 7.2)₂(final concentration 3mM) and UDP-glucose (final concentration 2.5 mM).

The reaction was allowed to proceed at 30 ℃ and samples were taken after 2, 4, 7 and 24 hours to determine conversion and initial rate by HPLC (CAD detection) using the analytical methods described above for conversion of rebaudioside D to rebaudioside M. Experiments were performed in parallel with the baseline clone round 1-Var 94. The conversion after 22h and the initial rate for this baseline clone were defined as 100%, and the normalized conversion and initial rate for the 2 nd round clone are depicted in the following table:

Mutations are labeled as follows: reference gene-original amino acid-position-new amino acid: for example, for the first round of directed evolution variant 94 from UGT76G1, the alanine mutation at position 33 to glycine was noted as round 1-Var 94(A33G)

Modeling of these results allows a ranking of the effect of each mutation to be obtained. The following mutations were identified as beneficial for activity: S42A, F46I, I190L, S274G, I295M, K303G, F31S, K316R, K393R, V394I, I407V, N409K, N409R, Q425E, Q432E, S447A, S456L.

Example 21

Directed evolution of UGT76G1 for conversion of rebaudioside D to rebaudioside X (round 3)

The most active clone from the second round of directed evolution of UGT76G1 (see example 20, second round _ UGT76G1var66 comprising mutation S42A _ F46I _ I407V) was selected as the baseline clone for the 3 rd round directed evolution. A list of 56 mutations was created, containing the different identified positive mutations from the second round and 30 new mutations obtained by the dna2.0protein gpstm strategy. This list of mutations was then used to design 92 variant genes, each containing 3 or 4 different mutations. After codon optimization for expression in e.coli, these genes were synthesized, subcloned into the pET30a + plasmid and used to transform e.coli BL21 (DE3) chemically competent cells. The obtained cells were grown in a petri dish on solid LB agar medium in the presence of kanamycin. Suitable colonies were selected and grown in liquid LB medium in test tubes. Glycerol was added to the suspension as a cryoprotectant and 400 μ Ι _ aliquots were stored at-20 ℃ and at-80 ℃.

These storage aliquots of E.coli BL21(DE3) containing the pET30a + _ UGT76G1var plasmid were thawed and added to LBGKP medium (20G/L Luria Broth Lennox; 50mM PIPES buffer pH 7.00; 50mM phosphate buffer pH 7.00; 2.5G/L glucose and 50mg/L kanamycin). This incubation was performed in 96 microtiter plates at 30 ℃ for 8h with shaking.

50 μ L of the above culture was inoculated into Overnight fast expression TB medium (Overnight Express) containing 60g/L^TM Instant TB medium)

10g/L glycerol and 50mg/L kanamycin in 3.95mL of production medium. The resulting culture was stirred at 20 ℃ in a 48-deep well plate. The culture produced significant growth and a good OD (600nm) was obtained. After 44 hours, cells were harvested by centrifugation and frozen.

By adding to thawed cells

Masterbatch

Lysis was performed and the lysate was recovered by centrifugation. Activity testing was performed with 100. mu.L of fresh lysate added to rebaudioside D (final concentration 0.5mM), MgCl, in 50mM phosphate buffer (pH 7.2)₂(final concentration 3mM) and UDP-glucose (final concentration 2.5 mM).

The reaction was allowed to proceed at 30 ℃ and samples were taken after 1, 2, 4, 6 and 22 hours to determine conversion and initial rate by HPLC (CAD detection) using the analytical methods described above for conversion of rebaudioside D to rebaudioside M. Experiments were performed in parallel with the baseline clone round 2-Var 66. The conversion after 22h and the initial rate for this baseline clone were defined as 100%, and the normalized conversion and initial rate for the 3 rd round clone are depicted in the following table:

Mutations are labeled as follows: reference gene-original amino acid-position-new amino acid: for example, for the second round of directed evolution variant 66 from UGT76G1, the isoleucine to leucine mutation at position 190 was labeled as round 2-Var 66(I190L)

Modeling of these results allows a ranking of the effect of each mutation to be obtained. The following mutations were identified as beneficial for activity:

I46L、I295M、S119A、S274G、K334R、F314S、K303G、K316R、 K393R、I190L、Q425E、Q432E、N138G、V394I、F182L、V407I、A272P、 V264C、E449D、A352G。

example 22

Conversion of rebaudioside A to rebaudioside I using UGT76G1

The reaction was carried out using UGT76G1-R1-F12 (also known as UGT76G1var94)

The total reaction volume was 40mL, with the following composition: 50mM potassium phosphate buffer pH 7.5, 3mM MgCl₂2.5mM UDP-glucose, 0.5mM rebaudioside A and 4mL UGT76G1-R1-F12 lysate (2.5U/mL). The reaction was run on an orbital shaker at 135rpm at 30 ℃. For sampling, 10. mu.L of 2N H was used₂SO₄And 115. mu.L of methanol/water (7/3) quench 125. mu.L of the reaction mixture. The samples were immediately centrifuged and maintained at 10 ℃ before analysis by LC-MS. An Agilent 1200 series HPLC system equipped with a binary pump (G1312B), an autosampler (G1367D), a thermostatted column box (G1316B), a DAD detector (G1315C) connected to an Agilent 6110A MSD and interfaced with "LC/MSD Chemstation" software was used.

Conditions of the apparatus

Mobile phase gradient procedure

The reaction characteristic curve is shown in fig. 5.

After 42 hours of reaction, 20mL of the reaction mixture was quenched with 20mL of ethanol and used for structural elucidation.

In a similar manner, the best clones for directed evolution of UGT76G1 for round 1 (UGT76G1-R1-F12), round 2 (UGT76G 1-R2-B9 identified above as "round 2-Var 66") and round 3 (UGT76G 1-R3-G3 identified above as "round 3-Var 21") and native UGT76G1 were tested for conversion of rebaudioside a to rebaudioside I. The results are shown in fig. 5.

Example 23

Isolation and characterization of rebaudioside I

Crude reaction sample.A sample for isolation of lot number crude CB-2977-198 was prepared with UGT76G1 according to example 22.

And (4) HPLC analysis.Preliminary HPLC analysis of the samples was performed using a Waters 2695Alliance system as follows: phenomenex Synergi Hydro-RP, 4.6X 250mm,4 μm (p/n 00G-4375-E0); column temperature: 55 ℃; mobile phase A: 0.0284% NH in water₄OAc and 0.0116% aqueous HOAc; mobile phase B: acetonitrile (MeCN); flow rate: 1.0 mL/min; injection volume: 10 μ L. Detection was performed by UV (210nm) and CAD.

Gradient:

time (min)	％A	％B
			0.0–8.5	75	25
10.0	71	29
			16.5	70	30
18.5–24.5	66	34
			26.5–29.0	48	52
31–37	30	70
			38	75	25

Separation was by HPLC.Using a Waters Atlantis dC18 (30X 100mm, 5 μm, p/n 186001375) column, isocratic flow 80:20 water/MeCN And (5) purifying under phase conditions. The flow rate was maintained at 45mL/min and the injection loading was 180 mg. The detector wavelength was set to 210 nm.

Fraction analysis was performed using: waters Atlantis dC18 (4.6X 150mm, 5 μm, p/n 186001342) column; mobile phase A: water; mobile phase B: MeCN; flow rate: 1 mL/min; isocratic mobile phase conditions: 75:25A/B for 30 minutes.

MS and MS/MS.MS and MS/MS data were generated using a Waters QT from a Micro mass spectrometer equipped with an electrospray ionization source. Samples were analyzed by negative ESI. Subjecting the sample to H₂MeCN (1:1) was diluted to a concentration of 0.25mg/mL and introduced by flow injection for MS data collection. Samples were further diluted to 0.01mg/mL to yield good s/n to tune for MS/MS and obtained by direct perfusion. The collision energy was set to 60V in order to obtain MS/MS data with increased fragment ion peaks due to the nature of the molecules

NMR。By dissolving about 1.0mg in 180. mu.L of pyridine-d₅+ TMS to prepare samples, and NMR data were obtained on a Bruker Avance 500 MHz instrument with a 2.5mm trans probe or a 5mm broadband probe. 13C and HMBC NMR data were obtained at the Rensselaer Polytechnic Institute using their Bruker Avance 600MHz and 800MHz instruments, respectively, with a 5mm cryoprobe (cryo-probe). ¹H and¹³c NMR spectra were referenced to TMS resonance (. delta.)_H0.00ppm and delta_C0.0ppm)。

The isolation of Reb I was carried out using a mixture of semisynthetic steviol glycosides (batch No. CB-2977-198). The material was analyzed by HPLC as described above. As shown in FIG. 6, at a retention time (t) of about 17 minutes_R) A Reb I peak was observed.

Results and discussion

The reb I peak was isolated from the reaction crude as described above. The separated fractions were combined and lyophilized. The purity of the final product was 91%, as confirmed by LC-CAD using the method described above. Approximately 1mg of reb I is provided for spectrophotometric and spectrometric analysis.

Mass spectrometry.ESI-TOF mass spectra obtained by perfusing reb I samples show at m/z 1127.4741 of [ M-H]^-Ions. [ M-H ]]^-Ion mass and molecular formula C expected for reb I₅₀H₇₉O₂₈(for C)₅₀H₇₉O₂₈The calculation is as follows: 1127.4758, error: 1.5ppm) was well consistent. MS data confirmed reb I to have a nominal mass of 1128 daltons and a molecular formula C₅₀H₈₀O₂₈。

MS/MS spectra of reb I (select [ M-H at M/z 1127.4)]^-Ion fragmentation) indicating the loss of two saccharide units at m/z 803.5301 but showing no additional fragmentation using a collision energy of 30V. When a higher collision energy (60V) was applied, no parent ion was observed, but the sequential loss of three sugar units at m/z 641.4488, 479.3897 and 317.3023 was observed from m/z 803.5301

NMR spectroscopy.Performing a series of NMR experiments, including¹H NMR、¹³C NMR、¹H-¹H COSY, HSQC-DEPT, HMBC NOESY, and perform 1D TOCSY to allow specification of reb I.

Of reb I obtained at 300K¹In the H NMR spectrum, one of the terminal protons is completely masked by the water resonance. Thus, obtaining samples at a lower temperature (292K)¹H NMR spectrum to remove the water resonance and at this temperature, the terminal protons are fully resolved. Thus, all other NMR data for reb I were obtained at 292K.

1D and 2D NMR data indicate that the central core of the glycoside is a diterpene. From at delta_HProton of methyl at 1.22 to delta_CThe HMBC association of the carbonyl group at 176.9 allows the designation of a tertiary methyl group (C-18) and one of C-19, and provides a starting point for the designation of the remaining aglycones. From the methyl proton (H-18) to delta_CAdditional HMBC associations of carbons at 38.5, 44.0, and 57.2 allow C-3, C-4, and C-5 to be specified.¹H-¹³Analysis of the C HSQC-DEPT data indicates that the peak value at delta_CCarbon at 38.5 is methylene and is at δ_CThe carbon at 57.2 is methine, which is designated C-3 and C-5, respectively. This is left at δ_CThe carbon at 44.0, which did not show a correlation in the HSQC-DEPT spectra, was designated as the quaternary carbon,and C-4. Assignment of C-3 (. delta.) to HSQC-DEPT data _H1.02 and 2.35) and C-5 (. delta.))_H1.03) of¹And H chemical shift. At one of the H-3 protons (delta)_H1.02) and at δ_HThe COSY association between protons at 1.44 allows one of the H-2 protons to be assigned, which in turn shows a delta to that assigned as H-1_HCorrelation of protons at 0.74. Then, based on additional COSY and HSQC-DEPT associations, the remainder for C-1 and C-2¹H and¹³c chemical shifts are assigned and are summarized in the table below.

¹H and¹³c NMR (500 and 150MHz, pyridine-d)₅) Designation of rebaudioside I aglycone

At delta_HThe other tertiary methyl singlet observed at 1.26 was shown to be associated with HMBC for C-1 and C-5 and was designated as H-20. The methyl proton shows a direct interaction with the quaternary carbons designated C-10 and C-9, respectively (delta)_C39.8) and methine carbon (. delta.)_C54.1) of the HMBC. Then, at H-5 (. delta.)_H1.03) and at δ_HThe COSY correlation between protons at 1.90 and 2.33 allows for the assignment of an H-6 proton, which H-6 proton in turn exhibits a delta with the proton assigned as H-7_HCorrelation of protons at 1.29 and 1.31. Then, the data on C-6 (. delta.) was determined from HSQC-DEPT_C22.2) and C-7 (. delta.))_C41.7) of¹³And C, chemical shift. At H-9 (delta)_H0.88) and at δ_HThe COSY correlation between protons at 1.67 and 1.70 allows for the assignment of an H-11 proton, which H-11 proton in turn exhibits a delta relationship with the proton assigned as H-12 proton _HCOSY correlation of protons at 1.98 and 2.28. HSQC-DEPT data was then used to assign C-11(δ)_C20.5) and C-12 (. delta.))_C37.3). At delta_H5.02 and 5.67 point viewsThe olefinic protons observed show a relationship with C-13 (. delta.)_C86.7) and is therefore designated as H-17 (delta via HSQC-DEPT)_C104.8). The methine proton H-9 shows δ with designations C-8, C-14 and C-15, respectively_CHMBC associations of carbons at 42.3, 44.3 and 47.6. The HSQC-DEPT data were used to assign at C-14 (delta)_H1.78 and 2.59) and C-15 (. delta.))_H2.04) of¹And H chemical shift. The additional HMBC associations from H-9 to C-11 and H-12 to C-9 further confirm the designation made above. Observed from H-14 and at δ_CHMBC association of the quaternary carbon at 154.0 allows for the assignment of C-16 to complete the assignment of the central core.

The correlation observed in the NOESY spectra was used to assign the relative stereochemistry of the central diterpene nucleus. In this NOESY spectrum, the NOE correlation observed between H-14 and H-20 indicates that H-14 and H-20 are on the same face of the ring. Similarly, NOE associations were observed between H-9 and H-5 and H-18. No NOE association between H-9 and H-14 was observed. Thus, NOESY data indicate that H-5, H-9 and H-18 are on opposite sides of the ring compared to H-14 and H-20. These data therefore indicate that the relative stereochemistry in the central nucleus is preserved during the glycosylation step.

In the case of the reb I, the first,¹H-¹³analysis of the C HSQC-DEPT data confirmed the presence of five terminal protons. At 292K at δ_H 6.14(δ_C 95.3)、5.57(δ_C 104.6)、5.38(δ_C 104.7)、5.29(δ_C105.0), and 5.06(δ)_C98.0) all five terminal protons are resolved in the spectrum obtained. In addition, all five terminal protons have large couplings (7.7Hz-8.2Hz), indicating that they have the β -configuration. At delta_HThe terminal proton observed at 6.14 showed a correlation with HMBC at C-19, indicating that it corresponds to Glc_IThe terminal protons of (1). Similarly, at δ_HThe terminal proton observed at 5.06 showed association with C-13 HMBC, allowing it to be designated as Glc_IIThe terminal protons of (1).

Glc_ITerminal proton (. delta.)_H6.14) exhibit a difference of delta_HDesignated Glc at 4.18_INature of H-2COSY association of children. COSY spectra do not allow for designation of H-3 or H-4 due to data overlap. Therefore, using Glc_ISelective irradiation of the terminal protons A series of 1D TOCSY experiments were performed at several different mixing times. Except that confirmation was made for Glc_IIn addition to the designation of H-2, the TOCSY data is shown at δ_HThe protons at 4.27, 4.25, and 3.93 are designated as H-3, H-4, and H-5, respectively. At δ in the TOCSY spectrum_HThe proton observed at 4.37 was designated Glc_IOne of the H-6 protons. Delta_HThe other H-6 methylene protons at 4.27 are based from H-5 to delta _H4.27 COSY association assigned. Using HSQC-DEPT data, specify about Glc_I C-2(δ_C 72.5)、 C-3(δ_C 89.4)、C-4(δ_C 69.2)、C-5(δ_C78.2-78.8), and C-6 (. delta.))_C61.7) of¹³And C, chemical shift. HMBC correlation of H-1 to C-3 and H-4 to C-6 further confirms the designation made above to complete Glc_IIs specified.

Of the four remaining unassigned glucose moieties, one was designated Glc based on HMBC associations_IA substituent at C-3 of (1). At delta_HThe terminal protons observed at 5.29 showed Glc interaction with_IHMBC association of C-3 and designated as Glc_VThe terminal protons of (1). Also observed is a change from Glc_IH-3 to Glc_VOpposite HMBC associations of anomeric carbons.

With respect to glycosides at C-19¹H and¹³a summary of the C chemical shifts is shown in the following table:

¹h and¹³c NMR (500 and 150MHz, pyridine-d)₅) Designation of rebaudioside I C-19 glycoside.

In the range of 78.2-78.8(78.16, 78.47, 78.50, 78.55 and 78.77)Five carbons resonate and thus chemical shifts cannot be specified unambiguously.

A summary of the key HMBC and COSY associations used to designate the C-19 glycoside region is provided below:

¹H-¹³c HMBC association

¹H-¹H COSY association

Glc_V(δ_H5.29) exhibit a proton exchange relationship with the terminal protons in delta_HDesignated Glc at 4.04_VCOSY association of protons of H-2. Glc was then assigned using HSQC-DEPT data _V C-2(δ_C75.3 or 75.5). COSY spectra do not allow for assignment of remaining protons due to data overlap. Therefore, using Glc_VSelective irradiation of the terminal protons A series of 1D TOCSY experiments were performed at several different mixing times. In addition to confirming the designation of GlcV H-2, the TOCSY data allows designation of Glc_V H-3(δ_H 4.27)、H-4(δ_H4.12), and H-5 (. delta.))_H4.05). At δ in the TOCSY spectrum_HThe proton observed at 4.56 was designated Glc_VOne of the H-6 protons. Delta_HThe other H-6 methylene protons at 4.26 are based on the atoms from H-5 to delta_H4.26 COSY association. Specifying information about Glc using HSQC-DEPT data_V C-3(δ_C 78.2-78.6)、C-4(δ_C71.5 or 71.6), C-5 (. delta.) and_C78.5 or 78.6) and C-6 (. delta.))_C62.3 or 62.4)¹³Chemical shift of C to complete Glc_VIs specified.

Glc was performed in a similar manner_IIIs specified. Glc_IIHeteropolar proton (. delta.)_H5.06) display and is assignedIs Glc_IIAt delta of H-2_HCOSY correlation of protons at 4.34 and further shown to correlate with_H 4.20(Glc_IICOSY correlation of protons at H-3), which shows correlation with delta_H 3.97(Glc_IIAdditional correlation of protons at H-4), which also shows correlation with delta_H3.80(Glc_IICOSY association of protons at H-5). H-5 shows a delta from that designated H-6_HAdditional COSY associations of protons at 4.18 and 4.49. Therefore, Glc is also used_IISelective irradiation of the terminal protons A series of 1D TOCSY experiments were performed at several different mixing times. The TOCSY data confirm the proton assignment described above. With regard to Glc _II C-2(δ_C 80.2)、C-3(δ_C87.5)、C-4(δ_C 70.1)、C-5 (δ_C77.6), and C-6 (. delta.))_C62.5) of¹³The assignment of C chemical shifts is based on HSQC-DEPT data. From Glc_IIH-3 to C-2 and C-4 and also from Glc_IIHMBC correlation of H-4 to C-3, C-5 and C-6 confirmed the designation made above, completing Glc_IIIs specified.

Based on HMBC association, the two remaining unspecified glucose moieties are specified as being at Glc_IIA substituent at C-2 and C-3 of (1). At delta_HThe terminal protons observed at 5.57 appear to interact with Glc_IIHMBC association of C-2 and designated as Glc_IIIThe terminal protons of (1). At delta_HThe terminal protons observed at 5.38 showed Glc interaction with_IIHMBC association of C-3 and designated as Glc_IVThe terminal protons of (1). It was also observed from Glc_IIH-2 to Glc_IIITerminal carbon of (3) and from Glc_IIH-3 to Glc_IVThe terminal carbons of (a) are HMBC related to each other.

Glc_III(δ_H5.57) exhibit a proton exchange relationship with the terminal protons in delta_HDesignated Glc at 4.21_IIICOSY association of protons of H-2. Glc was then assigned using HSQC-DEPT data_III C-2(δ_C76.3). COSY spectra do not allow for assignment of remaining protons due to data overlap. Therefore, using Glc_IIISelective irradiation of terminal protons in a series with several different mixing times1D TOCSY experiment. In addition to confirming the designation of GlcIII H-2, the TOCSY data allows designation of Glc_III H-3 (δ_H 4.27)、H-4(δ_H4.25) and H-5 (. delta.)) _H3.94). At δ in the TOCSY spectrum_H4.41 and delta_HThe proton observed at 4.53 was designated Glc_IIIH-6 proton. Assignment of C-3 (. delta.) using HSQC-DEPT data_C 78.2-78.6)、C-4(δ_C72.1)、C-5(δ_C78.2-78.8) and C-6 (. delta.))_C63.1) of¹³And C, chemical shift. From H-5 to delta_CHMBC correlation of carbon at 63.1 further confirms Glc_IIIAssignment of C-6, thus completing Glc_IIIIs specified.

Glc_IV(δ_H5.38) exhibit a proton exchange relationship with the terminal protons at delta_HDesignated Glc at 4.01_IVCOSY association of protons of H-2. Glc was then assigned using HSQC-DEPT data_IV C-2(δ_C75.3 or 75.5). COSY spectra do not allow for assignment of remaining protons due to data overlap. Therefore, using Glc_IVSelective irradiation of the terminal protons A series of 1D TOCSY experiments were performed at several different mixing times. Except that Glc was confirmed_IVIn addition to the designation of H-2, the 1D TOCSY data allows the designation of H-3(δ)_H 4.28)、H-4(δ_H 4.11)、H-5(δ_H4.13) and H-6 (. delta.))_H4.25 and 4.58). At delta_HThe proton at 4.25 also shows a correlation with delta_H4.58, further confirming that these protons belong to H-6. Assignment of C-3 (. delta.) using HSQC-DEPT data_C78.2-78.6)、C-4(δ_C 72.1)、C-5(δ_C78.2-78.6) and C-6 (. delta.) (delta.)_C62.3 or 62.4)¹³And C, chemical shift. HMBC correlation of H-4 to C-6 and H-5 to C-1 further confirmed Glc_IVAssignment of C-6, thus completing Glc_IVIs specified.

With respect to glycosides at C-13¹H and ¹³A summary of the C chemical shifts is shown below:

¹h and¹³c NMR (500 and 150MHz, pyridine-d)₅) Designation of rebaudioside I C-13 glycoside.

Five carbon resonances in the range 78.2-78.8(78.16, 78.47, 78.50, 78.55, and 78.77) and therefore chemical shifts cannot be specified explicitly.

A summary of the key HMBC and COSY associations used to designate the C-13 glycoside region is provided below:

¹H-¹³c HMBC association

¹H-¹H COSY association

NMR and MS analysis of rebaudioside I allowed the structure to be fully specified as (13- [ (2-O- β -D-glucopyranosyl-3-O- β -D-glucopyranosyl) oxy ] ent-kauri-16-en-19-oic acid (3-O- β -D-glucopyranosyl) ester ].

Example 24

Sensory characteristics of rebaudioside I

Rebaudioside I was evaluated to determine its sensory profile, including comparison to rebaudioside M.

Sample preparation. Samples were prepared in a water matrix at a concentration of 400 ppm. Analysis was performed to determine the actual concentration of the sample.

Method

Seven panelists participated in the rebaudioside I and rebaudioside M tests. Samples were provided at about 4 ℃. Panelists were instructed to drink 1 mouth sample (10mL), hold in the mouth for 5 seconds, spit out and evaluate taste attributes as described below. A 5 minute pause was set between each sample and panelists were instructed to wash their taste with at least 1 mouth of salt-free biscuit and 2 mouths of filtered water. The samples were randomized and each sample was presented in a repeating fashion over this period.

The attributes evaluated include: sweetness intensity (maximum sweetness level in the mouth over the course of 5 seconds); bitterness intensity (maximum bitterness level in the mouth during 5 seconds); overall maximum sweetness intensity (maximum sweetness intensity over a period of 1 minute from drinking one mouthful); overall maximum bitterness intensity (maximum bitterness intensity experienced from one mouthful to 1 minute); other intensities (intensity of any taste, aroma or mouthfeel (e.g., metal, plastic, licorice, etc.) in addition to sweetness and bitterness); sweetness linger intensity (sweetness intensity 1 minute after spitting out the sample); and bitter aftertaste intensity (bitter aftertaste intensity 1 minute after spitting out the sample).

Sweeteners were compared for each attribute using a 3-way ANOVA analysis and significance was determined at 95% CL. Fishers LSD was used to compare significant differences between mean scores.

Results

The sweetness intensity, overall maximum sweetness intensity, and sweetness linger intensity of rebaudioside I are lower.

Table 1: sensory attributes of rebaudioside I compared to rebaudioside M at 400ppm

Example 25: beverage formulations

Seasoning black tea: the taste characteristics of a flavoured zero calorie black tea beverage containing Reb a at a concentration of 275ppm were compared with a comparable flavoured zero calorie black tea beverage having Reb I at a concentration of 275 ppm. Beverages containing Reb I were identified as fresher aftertaste, with less sweetness linger and a fuller overall sweetness profile.

Enhanced water: the taste characteristics of a zero calorie enhanced water beverage containing Reb A at a concentration of 200ppm were compared to a comparable zero calorie enhanced water beverage containing Reb I at a concentration of 200 ppm. The Reb I containing beverage is fresher aftertaste with reduced sweetness linger and fuller overall sweetness taste quality.

Lemon acid flavored sparkling beverage: in zero caloriesSour lemonReb I levels were evaluated in flavored foaming beverage bases to determine the sweetness enhancing effect. Preparation ofSour lemonA sample of a flavored foaming beverage wherein the amount of Reb I is between 400 and 750ppm (in 50ppm increments) and the allulose is 3.5%. The taste of all samples was significantly better than the comparable Reb a containing formulations, resulting in a fresher profile with increased sweetness intensity and no negative aftertaste profile. The samples with 550ppm and 600ppm Reb I were found to be sweetened with 10.0Brix HFCS at the sweet taste levelSour lemonFlavored foaming beverage formulations are the closest.

Claims (10)

1. A method of preparing a rebaudioside I composition, comprising:

contacting a starting composition comprising rebaudioside a with a biocatalyst capable of converting rebaudioside a to rebaudioside I to provide a composition comprising rebaudioside I;

Wherein the biocatalyst is UGT;

wherein the UGT is a UGT76G1 variant; and is

Wherein the UGT76G1 variant is selected from the group consisting of:

UGT76G1-R1-F12, wherein the mutation points in UGT76G1-R1-F12 are: Q266E, P272A, R334K, G348P and L379G;

UGT76G1-R2-B9, which is UGT76G1-R1-F12 with additional mutation points S42A, F46I and I407V; and

UGT76G1-R3-G3, which is UGT76G1-R2-B9 with additional mutation points I46L, K303G and K393R.

2. The method of claim 1, wherein the UGT is provided in a form selected from pure form, crude lysate, or whole cell suspension.

3. The method of claim 1 or 2, wherein the UGT is provided microbiologically.

4. The method of claim 1, wherein rebaudioside a is provided in pure form, or in a steviol glycoside mixture or stevia extract containing at least 50% rebaudioside a by weight.

5. The method of claim 1, wherein the rebaudioside I composition comprises greater than 1% rebaudioside I by weight.

6. The method of claim 1, further comprising separating rebaudioside I to provide a separated rebaudioside I composition.

7. The method of claim 6, further comprising purifying the separated rebaudioside I composition to provide a highly purified rebaudioside I comprising greater than 80% rebaudioside I by weight.

8. A method of preparing a highly purified rebaudioside I composition comprising:

a. contacting a starting composition comprising rebaudioside a with a UGT76G1 variant group and UDP-glucose to form a composition comprising rebaudioside I, and concomitantly recycling UDP-glucose by providing sucrose synthase and sucrose;

wherein the UGT76G1 variant group is selected from the group consisting of:

UGT76G1-R1-F12, wherein the mutation points in UGT76G1-R1-F12 are: Q266E, P272A, R334K, G348P and L379G;

UGT76G1-R2-B9, which is UGT76G1-R1-F12 with additional mutation points S42A, F46I and I407V; and

UGT76G1-R3-G3, which is UGT76G1-R2-B9 with additional mutation points I46L, K303G and K393R;

b. separating rebaudioside I to form a separated rebaudioside I composition; and is

c. Purifying the separated rebaudioside I composition to provide a highly purified rebaudioside I composition; wherein the highly purified rebaudioside I comprises greater than 80% rebaudioside I by weight.

9. The method of claim 8, further comprising:

a. contacting a composition comprising stevioside with UGT76G1 and UDP-glucose to provide a composition comprising rebaudioside A, and

b. And separating the rebaudioside A.

10. The method of claim 8, further comprising:

a. contacting a composition comprising rebaudioside with UGT91D2 and UDP-glucose to provide a composition comprising stevioside; and is

b. Separating stevioside.

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